Biocatalytic Synthesis of Strained Carbocycles

ABSTRACT

Provided herein are methods for producing products containing strained carbocycles, such as cyclopropene moieties and/or bicyclobutane moieties. The methods include combining an alkyne and a carbene precursor in the presence of a heme protein, e.g., a cytochrome P450, under conditions sufficient to form the strained carbocycle. Reaction mixtures for producing strained carbocycles are also described, as well as whole-cell catalysts comprising heme proteins and variants thereof for forming cyclopropenes, bicyclobutanes, and related products.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional Pat. Appl.No. 62/474,435, filed on Mar. 21, 2017, and U.S. Provisional Pat. Appl.No. 62/583,073, filed on Nov. 8, 2017, which applications areincorporated herein by reference in their entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under Grant Nos.CBET1403077 and MCB1513007 awarded by the National Science Foundation.The government has certain rights in the invention.

REFERENCE TO SUBMISSION OF A SEQUENCE LISTING AS A TEXT FILE

The Sequence Listing written in fileSEQ_086544-1074284-020920US_ST25.txt created on Apr. 18, 2018, 62,516bytes, machine format IBM-PC, MS-Windows operating system, is herebyincorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Carbocycles serve as important structural motifs in different fields.(See, e.g., Chen, et al. Chem. Soc. Rev. 2012, 41, 4631; Williams, etal. Bioorg. Med. Chem. 2012, 20, 3710; Triola, et al. J. Org. Chem.2003, 68, 9924; Ando, et al. Bioorg. Med. Chem. 1999, 7, 571.) Thehigher ring strain energy of cyclopropenes, methylenecyclopropanes andbicyclobutanes, makes these compounds far more reactive than other ringsystems. (See, Wiberg. Angew. Chem. Int. Ed. 1986, 25, 312; Nakamura, etal. Chem. Rev. 2003, 103, 1295; Trost. Angew. Chem. Int. Ed. 1986, 25,1.) In synthetic chemistry, these highly strained rings have beeninvestigated as versatile building blocks with numerous derivatizationpossibilities; this has led to broad applications of thesethree-membered carbocycles in pharmaceutical development, chemicalbiology, material science, etc. (See, e.g., Marek, et al. Angew. Chem.Int. Ed. 2007, 46, 7364; Rubin, et al. Chem. Rev. 2007, 107, 3117; Fox,et al. Curr. Org. Chem. 2005, 9, 719; Parra, et al. J. Am. Chem. Soc.,2014, 136, 15833; Elling, et al. Chem. Commun. 2016, 52, 9097.) Forinstance, difunctionalization of cyclopropenes can introduce variousfunctional groups to the rings, which furnishes practical approaches tohighly functionalized three-membered ring structures in pharmaceuticalcompounds. (See, Tarwade, et al. J. Am. Chem. Soc. 2009, 131, 5382;O'Rourke, et al. Org. Lett. 2016, 18, 1250; Delaye, et al. Angew. Chem.Int. Ed. 2013, 52, 5333; Zhang, et al. Angew. Chem. Int. Ed. 2016, 55,714.) Cyclopropenes can undergo in vivo cycloaddition reactions andfunction as amenable bioorthogonal fluorescent tags for live cellimaging. (See, Patterson, et al. J. Am. Chem. Soc. 2012, 134, 18638; etal. J. Am. Chem. Soc. 2013, 135, 13680; et al. Angew. Chem. Int. Ed.2012, 51, 7476.) Besides, bicyclo[1.1.0]butane, is also very useful forconstructing a multitude of complicated structural scaffolds insynthetic chemistry. (See, Walczak, et al. Acc. Chem. Res. 2015, 48,1149; Wipf, et al. Angew. Chem. Int. Ed. 2006, 45, 4172; Gianatassio, etal. Science 2016, 351, 241; Wipf, et al. Am. Chem. Soc. 2003, 125,14694; Panish, et al. Angew. Chem. Int. Ed. 2016, 55, 4983.)

To fully realize the broad applicability of cyclopropenes andbicyclobutanes, it is highly desirable to develop practical andefficient methods for synthesizing these molecules with highselectivity. Transition metal-catalyzed carbene transfer to alkynesstands out as the most commonly used strategy to prepare cyclopropenesenantioselectively. (See, e.g., Doyle, et al. J. Am. Chem. Soc. 1994,116, 8492;. Lou, et al. J. Am. Chem. Soc. 2004, 126, 8916; Briones, etal. J. Am. Chem. Soc. 2010, 132, 17211; Uehara, et al. J. Am. Chem. Soc.2011, 133, 170; Goto, et al. Angew. Chem. int. Ed. 2011, 50, 6803; Cui,et al. J. Am. Chem. Soc. 2011, 133, 3304; Briones, et al. J. Am. Chem.Soc. 2012, 134, 11916.) Although synthetic chemists have exploitedvarious transition metal catalysts (e.g., rhodium, iridium, cobalt,copper and gold complexes) to achieve alkyne cyclopropenation, there arestill many challenges to be addressed: 1) most existing methods arelimited to conjugated carbene precursors with phenyl or cyano groups tostabilize the carbenoid intermediates to achieve goodenantio-/chemo-selectivity; 2) complicated chiral ligands or catalyticcomplexes require multiple-step preparation (4-15 steps); 3) thecatalytic efficiency is generally very low with <100 turnovers. Forbicyclobutane synthesis, a three-step sequence is commonly involved,which relies on the use of excess organolithium reagents, rendering itless practical for broad substrate scope and industrial production.(See, Dueker, et al. Tetrahedron Lett. 1985, 26, 3555; Rehm, et al. Eur.J. Org. Chem. 1999, 2079.)

Unlike synthetic catalysts, which require elaborate design,multiple-step preparation and optimization, natural enzymes aregenetically encoded and assembled in living cells, making them readilyaccessible and tunable with molecular biology techniques. (See, e.g.,Turner, et al. Nat. Chem. Biol. 2013, 9, 285; Kohler, et al. Chem.Commun. 2015, 51, 450.) Beyond this, enzymatic reactions commonlyoperate with high efficiency and selectivity (chemo-, regio-and/orstereo-) under mild conditions, offering significant advantages in termsof reduced environmental impact and greater cost-effectiveness. (See,Wohlgemuth. Curr. Opin. Biotechnol. 2010, 21, 713; Steen, et al. Nature2010, 463, 559; Peralta Yahya, et al. Nature 2012, 488, 320; Atsumi, etal. Nature 2008, 451, 86.) Due to the scarcity of cyclopropenes andbicyclobutanes in nature, no native enzymes have been discovered tocatalyze the construction of these highly strained three-memberedcarbocycles to date. (See, Schneider, et al. Proc. Natl. Acad. Sci.U.S.A. 2007, 104, 18941).

Over the last several years, the Arnold lab has been investigatingheme-dependent metalloenzymes for non-natural biocatalytic activities,including alkene cyclopropanation, carbenoid N—H insertion,aziridination, sulfimidation, and nitrenoid C—H insertion. See, e.g.,Hernandez, Kan, Arnold, et al. ACS Catalysis 2016, 6, 7810; Renata,Arnold, et al. Angew. Chem. Int. Ed. 2015, 54, 3351; Wang, Arnold, etal. Chem. Sci. 2014, 5, 598; Hyster & Arnold. Isr. J. Chem. 2015, 55,14; Coelho, Arnold, et al. Nat. Chem. Biol. 2013, 9, 485; Arnold. Rev.Biophys. 2015, 48, 404; Coelho, Arnold, et al. Science 2013, 339, 307;Kan, Chen, Arnold, et al. Science 2016, 354, 1048; Kan, Huang, Chen,Arnold, et al. Nature 2017, 552, 13. Now described herein is anefficient and practical protocol for synthesizing these highly strainedcarbocycles using hemoproteins engineered via directed evolution thatcatalyze carbene chemistry.

BRIEF SUMMARY OF THE INVENTION

Provided herein are methods for producing products containingcyclopropene moieties and/or bicyclobutane moieties. The methods includecombining an alkyne and a carbene precursor in the presence of a hemeprotein or a variant thereof under conditions sufficient to form thecyclopropene moiety and or the bicyclobutane moiety.

In some embodiments, the heme protein is a cytochrome P450, a globin, aprotoglobin, or a variant thereof. In some embodiments, the heme proteinis Bacillus megaterium cytochrome P450_(BM3) or a variant thereof. Insome embodiments, the carbene precursor is a diazo compound such as anα-diazoester.

Also provided are reaction mixtures for producing products containingcyclopropenes and bicyclobutanes, as well as whole-cell catalystscomprising heme proteins and variants thereof for forming such products.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of P450-catalyzed alkyne cyclopropenation. Leftbars=% yield; right bars=total turnover number (TTN).

FIG. 2 shows that P450 enzymes can be engineered to providecyclopropenation products enantioselectively.

FIG. 3 shows the P450-catalyzed bicyclobutanation of phenylacetylene.

FIG. 4 shows compounds with ring structures and calculated ring strains.The 4-membered carbocycles, bicyclo[1.1.0]but-1(3)-ene andtricyclo[1.1.0.02,4]butane (tetrahedrane) (with calculated strainenergy) have never been isolated, although their derivatives with bulkysubstituents might be synthesized in stable form under certainconditions.

FIG. 5A shows two major synthetic methods for forming bicyclobutanes.

FIG. 5B shows other synthetic methods for forming bicyclobutanes, withselected examples.

FIG. 5C. shows the only synthetic examples of asymmetric bicyclobutaneconstruction.

FIG. 6A shows examples of cyclopropane-containing natural products (ref.21, 62)

FIG. 6B shows examples of natural products that have been found tocontain cyclopropene fragments.

FIG. 6C shows thee only natural product that has been found to contain abicyclobutane fragment:

FIG. 7A shows the reaction of carbene transfer to an alkyne catalyzed byan engineered hemeprotein.

FIG. 7B shows the proposed catalytic cycle of carbene transfer tophenylacetylene to form cyclopropene and bicyclobutane structures.

FIG. 7C shows the screening of hemin and hemeprotein catalysts forbicyclobutane formation (BSA=bovine serum albumin).

FIG. 7D shows the X-ray crystal structure of P411-E10 (PDB ID: 5UCW)(35) and a view of its distal heme region. The P411 heme axial ligand isS400, and amino acid residues V78, L263 and S438 are shown as graysticks.

FIG. 7E shows the directed evolution of P411-E10 for bicyclobutaneformation (using phenylacetylene and EDA as substrates; numbers refer tototal turnovers to product (TTN) measured). Experiments were performedon analytical scale using suspensions of E. coli expressing P411-E10variants (OD₆₀₀=10−30), 10 mM phenylacetylene, 10 mM EDA, 5 vol % EtOH,M9-N buffer (pH 7.4) at room temperature under anaerobic conditions for6 h. Reactions performed in quadruplicate. TTN refers to the totaldesired product, as quantified by gas chromatography (GC), divided bytotal hemeprotein. Because bicyclobutane formation requires two carbenetransfers, the number of carbene transfers the hemeprotein catalyzes is2×TTN in these reactions.

FIG. 8 shows the time course of furan formation with P411-S1 I263Wvariant.

FIG. 9 shows the evolutionary trajectory of P411 variants forbicyclobutane formation.

FIG. 10 shows alkyne substrates used in protein evolution studies.

FIG. 11A shows the scope of P411-E10 V78F S438A-catalyzed bicyclobutaneformation. Standard conditions of preparative-scale reactions (0.1-0.2mmol scale, unless otherwise indicated): suspension of E. coli(OD₆₀₀=15-20) expressing P411 E10-V78F S438A, 1.0 equiv aromatic alkyne,2.0-4.0 equiv EDA, 10-15 mM D-glucose, 1-5 vol % EtOH, M9-N buffer (pH7.4) at room temperature under anaerobic conditions for 12 hours.Isolated yields. TTN determined based on isolated yields.

FIG. 11B shows the derivatization of bicyclobutane products. Top panel:copper-catalyzed click cyclization of 2 l with azide substrates. Bottomleft panel: esterification of 2 m with Mosher's acid. Bottom rightpanel: reduction of 2 k to diol with LiBH₄.

FIG. 12A shows the evolutionary trajectory of P411-P4 variants forstereodivergent cyclopropenation of aliphatic alkynes.

FIG. 12B shows the scope of P411-C6-catalyzed cyclopropene formation.Conditions of preparative-scale reactions (0.08-0.4 mmol scale):suspension of E. coli expressing P411-C6 or K10 (OD₆₀₀=10-32), 10-150 mMalkyne, 1.0-4.0 equiv EDA (6.0 equiv for 5m), 10-15 mM D-glucose, 1-5vol % EtOH, M9-N buffer (pH 7.4) at room temperature under anaerobicconditions for 12 hours. Isolated yields. TTN determined based onisolated yields and enantiomeric ratio (er) determined by chiral HPLC.

FIG. 12C shows the enzymatic cyclopropenation at mmol scale andderivatization of corresponding products. Top right: copper-catalyzedaddition to cyclopropene 6a for synthesizing a multi-substitutedcyclopropane. Bottom right: Diels-Alder reaction of cyclopropene 6h with2,3-diMe-buta-1,3-diene to form a fused ring system.

FIG. 13 shows the evolutionary trajectory of P411 variants for theformation of cyclopropene (S)-6a

FIG. 14 shows the evolutionary trajectory of P411 variants for theformation of cyclopropene (R)-6a

FIG. 15 shows the sites targeted in the evolution of P411-P4 for(S)-cyclopropene formation (PDB: 4WG2, P411-“I263F” (43)).

FIG. 16 shows the sites targeted in the directed evolution of P411-P4for (R)-cyclopropene formation (PDB: 4WG2, P411-“I263F” (43)).

DETAILED DESCRIPTION OF THE INVENTION I. SUMMARY

Demonstrated herein is a hemoprotein-catalyzed protocol for constructingstrained carobcycles such as cyclopropenes and bicyclobutanes. Describedherein is the first use of iron-based catalysts for alkynecyclopropenation and the first example of enantioselectivebicyclobutanation through carbene transfer to alkyne. By using directedevolution, it was possible to improve the reactivity of cytochrome P450sfor the desired construction of highly strained three-membered carbonrings. With the engineered enzymes, expressed and used in wholebacterial cells, chemoselective and stereodivergent cyclopropenation ofaliphatic alkynes was achieved with total turnover numbers (TTNs) up to3300 and enantiomeric excesses (ee's) up to >99.5%. Also provided hereinis a bicyclobutanation transformation sequence starting from aromaticalkynes with TTNs up to 1800. After hydrolysis, the bicyclobutaneproducts can be used to synthesize strained polyesters and otherproducts. The methods described herein can therefore be used to providea number of useful strained carbocycles. Carbocycles such ascyclopropenes and bicyclobutanes include several classes of compoundsincluding, but not limited to, natural products, pharmaceuticalcompounds, precursors for industrial products such as polymers,functional materials such as stimuli-responsive materials andself-healing materials, and labeling reagents for biological systems.

II. DEFINITIONS

Unless specifically indicated otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by thoseof ordinary skill in the art to which this invention belongs. Inaddition, any method or material similar or equivalent to a method ormaterial described herein can be used in the practice of the presentinvention. For purposes of the present invention, the following termsare defined.

The terms “a,” “an,” or “the” as used herein not only include aspectswith one member, but also include aspects with more than one member. Forinstance, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the reagent” includes reference to one or more reagentsknown to those skilled in the art, and so forth.

The terms “about” and “approximately” shall generally mean an acceptabledegree of error for the quantity measured given the nature or precisionof the measurements. Typical, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values. Alternatively, and particularly inbiological systems, the terms “about” and “approximately” may meanvalues that are within an order of magnitude, preferably within 5-foldand more preferably within 2-fold of a given value. Numerical quantitiesgiven herein are approximate unless stated otherwise, meaning that theterm “about” or “approximately” can be inferred when not expresslystated.

The terms “heme protein variant” and “heme enzyme variant” include anyheme-containing enzyme comprising at least one amino acid mutation withrespect to wild-type and also include any chimeric protein comprisingrecombined sequences or blocks of amino acids from two, three, or moredifferent heme-containing enzymes.

The term “whole cell catalyst” includes cells expressing heme-containingenzymes, wherein the whole cell catalyst displays cyclopropene formationactivity or bicyclobutane formation activity.

The term “carbene precursor” includes molecules that can be decomposedin the presence of metal (or enzyme) catalysts to form structures thatcontain at least one divalent carbon with two unshared valence shellelectrons (i.e., carbenes) and that can be transferred to acarbon-hydrogen bond, a carbon-carbon bond, a carbon-sulfur bond, acarbon-nitrogen bond, a carbon-boron bond, or a carbon-phosphorus bondto form various carbon ligated products. Examples of carbene precursorsinclude, but are not limited to, diazo reagents, diazirene reagents, andepoxide reagents.

As used herein, the term “anaerobic”, when used in reference to areaction, culture or growth condition, is intended to mean that theconcentration of oxygen is less than about 25 preferably less than about5 and even more preferably less than 1 μM. The term is also intended toinclude sealed chambers of liquid or solid medium maintained with anatmosphere of less than about 1% oxygen. Preferably, anaerobicconditions are achieved by sparging a reaction mixture with an inert gassuch as nitrogen or argon.

As used herein, the term “alkyl” refers to a straight or branched,saturated, aliphatic radical having the number of carbon atomsindicated. Alkyl can include any number of carbons, such as C₁₋₂, C₁₋₃,C₁₋₄, C₁₋₅, C₁₋₆, C₁₋₇, C₁₋₈, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄, C₃₋₅, C₃₋₆,C₄₋₅, C₄₋₆, and C₅₋₆. For example, C₁₋₆ alkyl includes, but is notlimited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl can refer toalkyl groups having up to 20 carbons atoms, such as, but not limited toheptyl, octyl, nonyl, decyl, etc. Alkyl groups can be unsubstituted orsubstituted. For example, “substituted alkyl” groups can be substitutedwith one or more moieties selected from halo, hydroxy, amino,alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.

As used herein, the term “alkenyl” refers to a straight chain orbranched hydrocarbon having at least 2 carbon atoms and at least onedouble bond. Alkenyl can include any number of carbons, such as C₂,C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₂₋₇, C₂₋₈, C₂₋₉, C₂₋₁₀, C₃, C₃₋₄, C₃₋₅, C₃₋₆,C₄, C₄₋₅, C₄₋₆, C₅, C₅₋₆, and C₆. Alkenyl groups can have any suitablenumber of double bonds, including, but not limited to, 1, 2, 3, 4, 5 ormore. Examples of alkenyl groups include, but are not limited to, vinyl(ethenyl), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl,butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl,1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl,1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl.Alkenyl groups can be unsubstituted or substituted. For example,“substituted alkenyl” groups can be substituted with one or moremoieties selected from halo, hydroxy, amino, alkylamino, alkoxy,haloalkyl, carboxy, amido, nitro, oxo, and cyano.

As used herein, the term “alkynyl” refers to either a straight chain orbranched hydrocarbon having at least 2 carbon atoms and at least onetriple bond. Alkynyl can include any number of carbons, such as C₂,C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₂₋₇, C₂₋₈, C₂₋₉, C₂₋₁₀, C₃, C₃₋₄, C₃₋₅, C₃₋₆,C₄, C₄₋₅, C₄₋₆, C₅, C₅₋₆, and C₆. Examples of alkynyl groups include,but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl,isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl,isopentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl,3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl,2,4-hexadiynyl, or 1,3,5-hexatriynyl. Alkynyl groups can beunsubstituted or substituted. For example, “substituted alkynyl” groupscan be substituted with one or more moieties selected from halo,hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro,oxo, and cyano.

As used herein, the term “aryl” refers to an aromatic carbon ring systemhaving any suitable number of ring atoms and any suitable number ofrings. Aryl groups can include any suitable number of carbon ring atoms,such as, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, as well asfrom 6 to 10, 6 to 12, or 6 to 14 ring members. Aryl groups can bemonocyclic, fused to form bicyclic or tricyclic groups, or linked by abond to form a biaryl group. Representative aryl groups include phenyl,naphthyl and biphenyl. Other aryl groups include benzyl, having amethylene linking group. Some aryl groups have from 6 to 12 ringmembers, such as phenyl, naphthyl or biphenyl. Other aryl groups havefrom 6 to 10 ring members, such as phenyl or naphthyl. Some other arylgroups have 6 ring members, such as phenyl. Aryl groups can beunsubstituted or substituted. For example, “substituted aryl” groups canbe substituted with one or more moieties selected from halo, hydroxy,amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, andcyano.

As used herein, the term “cycloalkyl” refers to a saturated or partiallyunsaturated, monocyclic, fused bicyclic or bridged polycyclic ringassembly containing from 3 to 12 ring atoms, or the number of atomsindicated. Cycloalkyl can include any number of carbons, such as C₃₋₆,C₄₋₆, C₅₋₆, C₃₋₈, C₄₋₈, C₅₋₈, and C₆₋₈. Saturated monocyclic cycloalkylrings include, for example, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic cycloalkylrings include, for example, norbornane, [2.2.2] bicyclooctane,decahydronaphthalene and adamantane. Cycloalkyl groups can also bepartially unsaturated, having one or more double or triple bonds in thering. Representative cycloalkyl groups that are partially unsaturatedinclude, but are not limited to, cyclobutene, cyclopentene, cyclohexene,cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene,cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene,and norbornadiene. Cycloalkyl groups can be unsubstituted orsubstituted. For example, “substituted cycloalkyl” groups can besubstituted with one or more moieties selected from halo, hydroxy,amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, andcyano.

As used herein, the term “heterocyclyl” refers to a saturated ringsystem having from 3 to 12 ring members and from 1 to 4 heteroatomsselected from N, 0 and S. Additional heteroatoms including, but notlimited to, B, Al, Si and P can also be present in a heterocycloalkylgroup. The heteroatoms can be oxidized to form moieties such as, but notlimited to, —S(O)— and —S(O)₂—. Heterocyclyl groups can include anynumber of ring atoms, such as, 3 to 6, 4 to 6, 5 to 6, 4 to 6, or 4 to 7ring members. Any suitable number of heteroatoms can be included in theheterocyclyl groups, such as 1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2to 3, 2 to 4, or 3 to 4. Examples of heterocyclyl groups include, butare not limited to, aziridine, azetidine, pyrrolidine, piperidine,azepane, azocane, quinuclidine, pyrazolidine, imidazolidine, piperazine(1,2-, 1,3- and 1,4-isomers), oxirane, oxetane, tetrahydrofuran, oxane(tetrahydropyran), oxepane, thiirane, thietane, thiolane(tetrahydrothiophene), thiane (tetrahydrothiopyran), oxazolidine,isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane,morpholine, thiomorpholine, dioxane, or dithiane. Heterocyclyl groupscan be unsubstituted or substituted. For example, “substitutedheterocyclyl” groups can be substituted with one or more moietiesselected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl,carboxy, amido, nitro, oxo, and cyano.

As used herein, the term “heteroaryl” refers to a monocyclic or fusedbicyclic or tricyclic aromatic ring assembly containing 5 to 16 ringatoms, where from 1 to 5 of the ring atoms are a heteroatom such as N, Oor S. Additional heteroatoms including, but not limited to, B, Al, Siand P can also be present in a heteroaryl group. The heteroatoms can beoxidized to form moieties such as, but not limited to, —S(O)— and—S(O)₂—. Heteroaryl groups can include any number of ring atoms, suchas, 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 3 to 9, 3 to10, 3 to 11, or 3 to 12 ring members. Any suitable number of heteroatomscan be included in the heteroaryl groups, such as 1, 2, 3, 4, or 5, or 1to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2 to 5, 3 to 4, or 3 to 5.Heteroaryl groups can have from 5 to 8 ring members and from 1 to 4heteroatoms, or from 5 to 8 ring members and from 1 to 3 heteroatoms, orfrom 5 to 6 ring members and from 1 to 4 heteroatoms, or from 5 to 6ring members and from 1 to 3 heteroatoms. Examples of heteroaryl groupsinclude, but are not limited to, pyrrole, pyridine, imidazole, pyrazole,triazole, tetrazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-,1,2,4- and 1,3,5-isomers), thiophene, furan, thiazole, isothiazole,oxazole, and isoxazole. Heteroaryl groups can be unsubstituted orsubstituted. For example, “substituted heteroaryl” groups can besubstituted with one or more moieties selected from halo, hydroxy,amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, andcyano.

As used herein, the term “alkoxy” refers to an alkyl group having anoxygen atom that connects the alkyl group to the point of attachment:i.e., alkyl-O—. As for alkyl group, alkoxy groups can have any suitablenumber of carbon atoms, such as C₁₋₆ or C₁₋₄. Alkoxy groups include, forexample, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy,iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc. Alkoxy groupscan be unsubstituted or substituted. For example, “substituted alkoxy”groups can be substituted with one or more moieties selected from halo,hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro,oxo, and cyano.

As used herein, the term “alkylthio” refers to an alkyl group having asulfur atom that connects the alkyl group to the point of attachment:i.e., alkyl-S—. As for alkyl groups, alkylthio groups can have anysuitable number of carbon atoms, such as C₁₋₆ or C₁₋₄. Alkylthio groupsinclude, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy,2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc.groups can be unsubstituted or substituted. For example, “substitutedalkylthio” groups can be substituted with one or more moieties selectedfrom halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy,amido, nitro, oxo, and cyano.

As used herein, the term “heteroalkyl” refers to an alkyl group havingone or more non-adjacent methylene (i.e., CH₂) units that is replaced byO, S, or NH. A carbon atom is the point of attachment for theheteroalkyl group to the remainder of the molecule, but the methylenereplacement can occur at any other point along the carbon backbone. Inthe case of oxygen for example, replacement of CH₂ can occur in themiddle of an alkyl group (e.g., in the middle of a propyl group, formingmethoxymethyl with the formula CH₃OCH₂—) or at the end of the alkylgroup (e.g., at the end of the propyl group, forming hydroxyethyl withthe formula HOCH₂CH₂—).

As used herein, the terms “halo” and “halogen” refer to fluorine,chlorine, bromine and iodine.

As used herein, the term “haloalkyl” refers to an alkyl moiety asdefined above substituted with at least one halogen atom.

As used herein, the term “alkylsilyl” refers to a moiety SiR₃, whereinat least one R group is alkyl and the other R groups are H or alkyl. Thealkyl groups can be substituted with one more halogen atoms.

As used herein, the term “acyl” refers to a moiety —C(O)R, wherein R isan alkyl group.

As used herein, the term “oxo” refers to an oxygen atom that isdouble-bonded to a compound (i.e., O═).

As used herein, the term “carboxy” refers to a moiety —C(O)OH. Thecarboxy moiety can be ionized to form the carboxylate anion. “Alkylcarboxylate” refers to a moiety —C(O)OR, wherein R is an alkyl group asdefined herein.

As used herein, the term “amino” refers to a moiety —NR3, wherein each Rgroup is H or alkyl.

As used herein, the term “amido” refers to a moiety —NRC(O)R or—C(O)NR₂, wherein each R group is H or alkyl.

As used herein, the term “phosphine” refers to a moiety —PR₃, whereineach R group is H, alkyl, cycloalkyl, aryl, or heterocyclyl.

The terms “protein,” “peptide,” and “polypeptide” are usedinterchangeably herein to refer to a polymer of amino acid residues, oran assembly of multiple polymers of amino acid residues. The terms applyto amino acid polymers in which one or more amino acid residues are anartificial chemical mimic of a corresponding naturally occurring aminoacid, as well as to naturally occurring amino acid polymers andnon-naturally occurring amino acid polymers.

The term “amino acid” includes naturally-occurring α-amino acids andtheir stereoisomers, as well as unnatural (non-naturally occurring)amino acids and their stereoisomers. “Stereoisomers” of amino acidsrefers to mirror image isomers of the amino acids, such as L-amino acidsor D-amino acids. For example, a stereoisomer of a naturally-occurringamino acid refers to the mirror image isomer of the naturally-occurringamino acid, i.e., the D-amino acid.

Naturally-occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, y-carboxyglutamate and O-phosphoserine.Naturally-occurring α-amino acids include, without limitation, alanine(Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu),phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile),arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met),asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser),threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), andcombinations thereof. Stereoisomers of naturally-occurring α-amino acidsinclude, without limitation, D-alanine (D-Ala), D-cysteine (D-Cys),D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine(D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg),D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine(D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser),D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine(D-Tyr), and combinations thereof.

Unnatural (non-naturally occurring) amino acids include, withoutlimitation, amino acid analogs, amino acid mimetics, synthetic aminoacids, N-substituted glycines, and N-methyl amino acids in either the L-or D-configuration that function in a manner similar to thenaturally-occurring amino acids. For example, “amino acid analogs” areunnatural amino acids that have the same basic chemical structure asnaturally-occurring amino acids, i.e., an a carbon that is bound to ahydrogen, a carboxyl group, an amino group, but have modified R (i.e.,side-chain) groups or modified peptide backbones, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. “Aminoacid mimetics” refer to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunctions in a manner similar to a naturally-occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. For example, an L-aminoacid may be represented herein by its commonly known three letter symbol(e.g., Arg for L-arginine) or by an upper-case one-letter amino acidsymbol (e.g., R for L-arginine). A D-amino acid may be representedherein by its commonly known three letter symbol (e.g., D-Arg forD-arginine) or by a lower-case one-letter amino acid symbol (e.g., r forD-arginine).

With respect to amino acid sequences, one of skill in the art willrecognize that individual substitutions, additions, or deletions to apeptide, polypeptide, or protein sequence which alters, adds, or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. The chemically similar amino acid includes, withoutlimitation, a naturally-occurring amino acid such as an L-amino acid, astereoisomer of a naturally occurring amino acid such as a D-amino acid,and an unnatural amino acid such as an amino acid analog, amino acidmimetic, synthetic amino acid, N-substituted glycine, and N-methyl aminoacid.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. For example, substitutions may be madewherein an aliphatic amino acid (e.g., G, A, I, L, or V) is substitutedwith another member of the group. Similarly, an aliphaticpolar-uncharged group such as C, S, T, M, N, or Q, may be substitutedwith another member of the group; and basic residues, e.g., K, R, or H,may be substituted for one another. In some embodiments, an amino acidwith an acidic side chain, e.g., E or D, may be substituted with itsuncharged counterpart, e.g., Q or N, respectively; or vice versa. Eachof the following eight groups contains other exemplary amino acids thatare conservative substitutions for one another:

-   1) Alanine (A), Glycine (G);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);-   7) Serine (S), Threonine (T); and-   8) Cysteine (C), Methionine (M)-   (see, e.g., Creighton, Proteins, 1993).

The term “oligonucleotide,” “nucleic acid,” “nucleotide,” or“polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleicacids (RNA) and polymers thereof in either single-, double- ormulti-stranded form. The term includes, but is not limited to, single-,double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNAhybrids, or a polymer comprising purine and/or pyrimidine bases or othernatural, chemically modified, biochemically modified, non-natural,synthetic or derivatized nucleotide bases. Unless specifically limited,the term encompasses nucleic acids containing known analogs of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), orthologs, andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res. 19:5081 (1991), Ohtsuka etal., J. Biol. Chem. 260:2605-2608 (1985), and Rossolini et al., Mol.Cell. Probes 8:91-98 (1994)). The term nucleic acid is usedinterchangeably with gene, cDNA, and mRNA encoded by a gene.

The term “site-directed mutagenesis” refers to various methods in whichspecific changes are intentionally made introduced into a nucleotidesequence (i.e., specific nucleotide changes are introduced atpre-determined locations). Known methods of performing site-directedmutagenesis include, but are not limited to, PCR site-directedmutagenesis, cassette mutagenesis, whole plasmid mutagenesis, andKunkel's method.

The term “site-saturation mutagenesis,” also known as “saturationmutagenesis,” refers to a method of introducing random mutations atpredetermined locations with a nucleotide sequence, and is a methodcommonly used in the context of directed evolution (e.g., theoptimization of proteins (e.g., in order to enhance activity, stability,and/or stability), metabolic pathways, and genomes). In site-saturationmutagenesis, artificial gene sequences are synthesized using one or moreprimers that contain degenerate codons; these degenerate codonsintroduce variability into the position(s) being optimized. Each of thethree positions within a degenerate codon encodes a base such as adenine(A), cytosine (C), thymine (T), or guanine (G), or encodes a degenerateposition such as K (which can be G or T), M (which can be A or C), R(which can be A or G), S (which can be C or G), W (which can be A or T),Y (which can be C or T), B (which can be C, G, or T), D (which can be A,G, or T), H (which can be A, C, or T), V (which can be A, C, or G), or N(which can be A, C, G, or T). Thus, as a non-limiting example, thedegenerate codon NDT encodes an A, C, G, or T at the first position, anA, G, or T at the second position, and a T at the third position. Thisparticular combination of 12 codons represents 12 amino acids (Phe, Leu,Ile, Val, Tyr, His, Asn, Asp, Cys, Arg, Ser, and Gly). As anothernon-limiting example, the degenerate codon VHG encodes an A, C, or G atthe first position, an A, C, or T at the second position, and G at thethird position. This particular combination of 9 codons represents 8amino acids (Lys, Thr, Met, Glu, Pro, Leu, Ala, and Val). As anothernon-limiting example, the “fully randomized” degenerate codon NNNincludes all 64 codons and represents all 20 naturally-occurring aminoacids.

In some instances, a mixture of degenerate primers is used. A mixture ofdegenerate primers can contain any number of different degenerateprimers in any ratio. As a non-limiting example, a mixture of primerscontaining the NDT, VHG, and TGG primers can be used. Such a mixture cancontain, for example, an amount of each primer in a 12:9:1 ratio (e.g.,a NDT:VHG:TGG ratio of 12:9:1). Based on various considerations,non-limiting examples being desired redundancy, the desired presence ofstop codons, and/or desired amino acid characteristics (e.g., thepresence of nonpolar residues, charged residues, or small side chainresidues), different combinations of degenerate primers can be used.Considerations and methods for choosing optimal combinations ofdegenerate primers will be known to one of skill in the art.

The term “nucleotide sequence encoding a peptide” means the segment ofDNA involved in producing a peptide chain. The term can include regionspreceding and following the coding region (leader and trailer) involvedin the transcription/translation of a gene product and the regulation ofthe transcription/translation, as well as intervening sequences(introns) between individual coding segments (exons).

The term “homolog,” as used herein with respect to an original enzyme orgene of a first family or species, refers to distinct enzymes or genesof a second family or species which are determined by functional,structural or genomic analyses to be an enzyme or gene of the secondfamily or species which corresponds to the original enzyme or gene ofthe first family or species. Homologs most often have functional,structural, or genomic similarities. Techniques are known by whichhomologs of an enzyme or gene can readily be cloned using genetic probesand PCR. Identity of cloned sequences as homolog can be confirmed usingfunctional assays and/or by genomic mapping of the genes.

A protein has “homology” or is “homologous” to a second protein if theamino acid sequence encoded by a gene has a similar amino acid sequenceto that of the second gene. Alternatively, a protein has homology to asecond protein if the two proteins have “similar” amino acid sequences.Thus, the term “homologous proteins” is intended to mean that the twoproteins have similar amino acid sequences. In particular embodiments,the homology between two proteins is indicative of its shared ancestry,related by evolution.

III. DESCRIPTION OF THE EMBODIMENTS

Provided herein are methods for the formation of cyclopropenes andbicyclobutanes. In certain aspects, the present invention provides amethod for producing a cyclopropene product in the presence of analiphatic alkyne, a diazo substrate, and a cytochrome P450 enzymeaccording to Equation 1:

wherein

R¹⁰ is selected from the group consisting of H and CR¹R²;

each R¹⁰ is independently selected from the group consisting of H, C₁₋₁₈alkyl, C₁₋₁₈ haloalkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, (C₃₋₁₀)cycloalkyl)-L¹-, (C₆₋₁₀) aryl)-L¹-, (5- to 10-membered heteroaryl)-L¹-,(5- to 10-membered heterocyclyl)-L¹-, cyano, halo, nitro, N(R⁸)₂,B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷, C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂,C(O)NR⁷OR⁸, C(O)C(O)OR⁷, and P(O)(OR⁷)₂;

C₁₋₁₈ alkyl, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, 5- to 10-membered heteroaryl,and 5- to 10-membered heterocyclyl in R¹ are optionally andindependently substituted with one or more R^(1a);

each R^(1a) is independently selected from the group consisting ofhalogen, cyano, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ heteroalkyl, C₂₋₆alkynyl, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, and 5- to 10-memberedheterocyclyl;

L¹ is selected from the group consisting of a bond and C₁₋₂₀ alkylene;

when L¹ is C₂₋₂₀ alkylene, one or more non-adjacent CH₂ groups areoptionally and independently replaced with O, S, or NH;

when L¹ is C₃₋₂₀ alkylene, one or more pairs of adjacent CH₂ groups areoptionally and independently replaced with C(O)O or C(O)NH;

each R² is independently selected from the group consisting of H, C₁₋₁₈alkyl, C₁₋₁₈ haloalkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, (C₃₋₁₀cycloalkyl)-L²-, (C₆₋₁₀ aryl)-L²-, (5- to 10-membered heteroaryl) L²-,(5- to 10-membered heterocyclyl)-L²-, cyano, halo, nitro, N(R⁸)₂,B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷, C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂,C(O)NR⁷OR⁸, C(O)C(O)OR⁷, and P(O)(OR⁷)₂;

C₁₋₁₈ alkyl, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, 5- to 10-membered heteroaryl,and 5- to 10-membered heterocyclyl in R² are optionally andindependently substituted with one or more R^(2a);

each R^(2a) is independently selected from the group consisting ofhalogen, cyano, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ heteroalkyl, C₂₋₆alkynyl, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, and 5- to 10-memberedheterocyclyl;

L² is selected from the group consisting of a bond and C₁₋₂₀ alkylene;

when L² is C₂₋₂₀ alkylene, one or more non-adjacent CH₂ groups areoptionally and independently replaced with O, S, or NH;

when L² is C₃₋₂₀ alkylene, one or more pairs of adjacent CH₂ groups areoptionally and independently replaced with C(O)O or C(O)NH;

R³ and R⁴ are independently selected from the group consisting of H,optionally substituted C₁₋₁₈ alkyl, optionally substituted C₁₋₁₈ alkoxy,C₁₋₁₈ haloalkyl (e.g., C₁₋₁₈ polyfluoroalkyl), C₂₋₁₈ alkenyl, C₂₋₁₈alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted 5- to10-membered heteroaryl, optionally substituted 5- to 10-memberedheterocyclyl, cyano, halo, nitro, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷,C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂, C(O)NR⁷OR⁸, C(O)C(O)OR⁷ andP(O)(OR⁷)₂; and each R⁷, R⁸, and R⁹ is independently selected from thegroup consisting of H, optionally substituted C₁₋₁₈ alkyl, C₂₋₁₈alkenyl, C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl, optionallysubstituted 5- to 10-membered heteroaryl, and optionally substituted 5-to 10-membered heterocyclyl.

In some embodiments, R¹ and R² are independently selected from the groupconsisting of H, optionally substituted C₁₋₁₈ alkyl, C₁₋₁₈polyfluoroalkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, optionally substitutedC₆₋₁₀ aryl, optionally substituted 6- to 10-membered heteroaryl,optionally substituted 6- to 10-membered heterocyclyl, cyano, halo,nitro, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷, C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷,C(O)ON(R⁷)₂, C(O)NR⁷OR⁸, C(O)C(O)OR⁷ and P(O)(OR⁷)₂.

In some embodiments, R³ and R⁴ are independently selected from the groupconsisting of H, optionally substituted C₁₋₁₈ alkyl, C₁₋₁₈polyfluoroalkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, optionally substitutedC₆₋₁₀ aryl, optionally substituted 6- to 10-membered heteroaryl,optionally substituted 6- to 10-membered heterocyclyl, cyano, halo,nitro, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷, C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷,C(O)ON(R⁷)₂, C(O)NR⁷OR⁸, C(O)C(O)OR⁷ and P(O)(OR⁷)₂.

In certain aspects, the present invention provides a method forproducing a bicyclobutane product in the presence of an alkyne, a diazosubstrate, and a hemoprotein according to Equation 2:

wherein

R¹¹ is selected from the group consisting of H, C₁₋₁₈ alkyl, C₁₋₁₈haloalkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, (C₃₋₁₀ cycloalkyl)-L¹¹-, (C₆₋₁₀aryl)-L¹¹-, (5- to 10-membered heteroaryl)-L¹¹-, (5- to 10-memberedheterocyclyl)-L¹¹-, cyano, halo, nitro, N(R¹⁸)₂, B(R¹⁹)₂, Si(R¹⁹)₃,C(O)OR¹⁷, C(O)SR¹⁷, C(O)N(R¹⁷)₂, C(O)R¹⁷, C(O)ON(R¹⁷)₂, C(O)NR¹⁷OR¹⁸,C(O)C(O)OR¹⁷, and P(O)(OR¹⁷)₂;

C₁₋₁₈ alkyl, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, 5- to 10-membered heteroaryl,and 5- to 10-membered heterocyclyl in R¹¹ are optionally andindependently substituted with one or more R^(11a);

each R^(11a) is independently selected from the group consisting ofhalogen, cyano, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ heteroalkyl, C₂₋₆alkynyl, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, and 5- to 10-memberedheterocyclyl;

L¹¹- is selected from the group consisting of a bond and C₁₋₂₀ alkylene;when L¹¹ is C₂₋₂₀ alkylene, one or more non-adjacent CH₂ groups areoptionally and independently replaced with O, S, or NH;

when L¹¹ is C₃₋₂₀ alkylene, one or more pairs of adjacent CH₂ groups areoptionally and independently replaced with C(O)O or C(O)NH;

R¹² is selected from the group consisting of H, C₁₋₁₈ alkyl, C₁₋₁₈haloalkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, (C₃₋₁₀ cycloalkyl)-L¹²-, (C₆₋₁₀aryl)-L¹²-, (5- to 10-membered heteroaryl) L¹²-, (5- to 10-memberedheterocyclyl)-L¹²-, cyano, halo, nitro, N(R¹⁸)₂, B(R¹⁹)₂, Si(R¹⁹)₃,C(O)OR¹⁷, C(O)SR¹⁷, C(O)N(R¹⁷)₂, C(O)R¹⁷, C(O)ON(R¹⁷)₂, C(O)NR¹⁷OR¹⁸,C(O)C(O)OR¹⁷, and P(O)(OR¹⁷)₂;

C₁₋₁₈ alkyl, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, 5- to 10-membered heteroaryl,and 5- to 10-membered heterocyclyl in le² are optionally andindependently substituted with one or more R^(12a);

each R^(12a) is independently selected from the group consisting ofhalogen, cyano, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ heteroalkyl, C₂₋₆alkynyl, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, and 5- to 10-memberedheterocyclyl;

L¹² is selected from the group consisting of a bond and C₁₋₂₀ alkylene;

when L¹² is C₂₋₂₀ alkylene, one or more non-adjacent CH₂ groups areoptionally and independently replaced with O, S, or NH;

when L¹² is C₃₋₂₀ alkylene, one or more pairs of adjacent CH₂ groups areoptionally and independently replaced with C(O)O or C(O)NH;

R¹³ and R¹⁴ are independently selected from the group consisting of H,optionally substituted C₁₋₁₈ alkyl, optionally substituted C₁₋₁₈ alkoxy,C₁₋₁₈ haloalkyl (e.g., C₁₋₁₈ polyfluoroalkyl), C₂₋₁₈ alkenyl, C₂₋₁₈alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted 5- to10-membered heteroaryl, optionally substituted 5- to 10-memberedheterocyclyl, cyano, halo, nitro, N(R¹⁸)₂, B(R¹⁹)₂, Si(R¹⁹)₃, C(O)OR¹⁷,C(O)SR¹⁷, C(O)N(R¹⁷)₂, C(O)R¹⁷, C(O)ON(R¹⁷)₂, C(O)NR¹⁷OR¹⁸, C(O)C(O)OR¹⁷and P(O)(OR¹⁷)₂; and each R¹⁷, R¹⁸, and R¹⁹ is independently selectedfrom the group consisting of H, optionally substituted C₁₋₁₈ alkyl,C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl,optionally substituted 5- to 10-membered heteroaryl, and optionallysubstituted 5- to 10-membered heterocyclyl.

In some embodiments, R¹¹ and R¹² are independently selected from thegroup consisting of H, optionally substituted C₁₋₁₈ alkyl, C₁₋₁₈haloalkyl (e.g., C₁₋₁₈ polyfluoroalkyl), C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl,optionally substituted C₆₋₁₀ aryl, optionally substituted 5- to10-membered heteroaryl, optionally substituted 5- to 10-memberedheterocyclyl, cyano, halo, nitro, N(R¹⁸)₂, B(R¹⁹)₂, Si(R¹⁹)₃, C(O)OR¹⁷,C(O)SR¹⁷, C(O)N(R¹⁷)₂, C(O)R¹⁷, C(O)ON(R¹⁷)₂, C(O)NR¹⁷OR¹⁸, C(O)C(O)OR¹⁷and P(O)(OR¹⁷)₂.

In some embodiments, R¹³ and R¹⁴ are independently selected from thegroup consisting of H, optionally substituted C₁₋₁₈ alkyl, optionallysubstituted C₁₋₁₈ alkoxy, C₁₋₁₈ haloalkyl (e.g., C₁₋₁₈ polyfluoroalkyl),C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl,optionally substituted 5- to 10-membered heteroaryl, optionallysubstituted 5- to 10-membered heterocyclyl, cyano, halo, nitro, N(R¹⁸)₂,B(R¹⁹)₂, Si(R¹⁹)₃, C(O)OR¹⁷, C(O)SR¹⁷, C(O)N(R¹⁷)₂, C(O)R¹⁷,C(O)ON(R¹⁷)₂, C(O)NR¹⁷OR¹⁸, C(O)C(O)OR¹⁷ and P(O)(OR¹⁷)₂.

This method produces products of Formula III and Formula VI, instereochemically pure form or as a mixture of stereochemically isomericforms. Exemplary catalysts used in this method include hemoproteins ofthe sort described in US 2014/0242647 A1.

A. Heme Proteins

The terms “heme protein” and “heme enzyme” are used herein to includeany member of a group of proteins containing heme as a prosthetic group.Non-limiting examples of heme proteins include globins, cytochromes,oxidoreductases, any other protein containing a heme as a prostheticgroup, and combinations thereof. Heme-containing globins include, butare not limited to, hemoglobin, myoglobin, and combinations thereof.Heme-containing cytochromes include, but are not limited to, cytochromeP450, cytochrome b, cytochrome c1, cytochrome c, and combinationsthereof. Heme-containing oxidoreductases include, but are not limitedto, catalases, oxidases, oxygenases, haloperoxidases, peroxidases, andcombinations thereof. In some instances, the globin protein is fromMethylacidiphilum infernorum. In some other instances, the cytochromeP450 protein is a cytochrome P450 BM3 (CYP102A1) protein. Exemplarycatalysts used in this method include, but are not limited to, hemeproteins of the sort described in US 20140242647 A1.

In some embodiments, the heme protein is a member of one of the enzymeclasses set forth in Table 1. In other embodiments, the heme protein isa variant or homolog of a member of one of the enzyme classes set forthin Table 1. In yet other embodiments, the heme protein comprises orconsists of the heme domain of a member of one of the enzyme classes setforth in Table 1 or a fragment thereof (e.g., a truncated heme domain)that is capable of carrying out the carbene insertion reactionsdescribed herein.

TABLE 1 Heme enzymes identified by their enzyme classification number(EC number) and classification name EC Number Name 1.1.2.3 L-lactatedehydrogenase 1.1.2.6 polyvinyl alcohol dehydrogenase (cytochrome)1.1.2.7 methanol dehydrogenase (cytochrome c) 1.1.5.5 alcoholdehydrogenase (quinone) 1.1.5.6 formate dehydrogenase-N: 1.1.9.1 alcoholdehydrogenase (azurin): 1.1.99.3 gluconate 2-dehydrogenase (acceptor)1.1.99.11 fructose 5-dehydrogenase 1.1.99.18 cellobiose dehydrogenase(acceptor) 1.1.99.20 alkan-1-ol dehydrogenase (acceptor) 1.2.1.70glutamyl-tRNA reductase 1.2.3.7 indole-3-acetaldehyde oxidase 1.2.99.3aldehyde dehydrogenase (pyrroloquinoline-quinone) 1.3.1.6 fumaratereductase (NADH): 1.3.5.1 succinate dehydrogenase (ubiquinone) 1.3.5.4fumarate reductase (menaquinone) 1.3.99.1 succinate dehydrogenase1.4.9.1 methylamine dehydrogenase (amicyanin) 1.4.9.2. aralkylaminedehydrogenase (azurin) 1.5.1.20 methylenetetrahydrofolate reductase[NAD(P)H] 1.5.99.6 spermidine dehydrogenase 1.6.3.1 NAD(P)H oxidase1.7.1.1 nitrate reductase (NADH) 1.7.1.2 Nitrate reductase [NAD(P)H]1.7.1.3 nitrate reductase (NADPH) 1.7.1.4 nitrite reductase [NAD(P)H]1.7.1.14 nitric oxide reductase [NAD(P), nitrous oxide-forming] 1.7.2.1nitrite reductase (NO-forming) 1.7.2.2 nitrite reductase (cytochrome;ammonia-forming) 1.7.2.3 trimethylamine-N-oxide reductase (cytochrome c)1.7.2.5 nitric oxide reductase (cytochrome c) 1.7.2.6 hydroxylaminedehydrogenase 1.7.3.6 hydroxylamine oxidase (cytochrome) 1.7.5.1 nitratereductase (quinone) 1.7.5.2 nitric oxide reductase (menaquinol) 1.7.6.1nitrite dismutase 1.7.7.1 ferredoxin-nitrite reductase 1.7.7.2ferredoxin-nitrate reductase 1.7.99.4 nitrate reductase 1.7.99.8hydrazine oxidoreductase 1.8.1.2 sulfite reductase (NADPH) 1.8.2.1sulfite dehydrogenase 1.8.2.2 thiosulfate dehydrogenase 1.8.2.3sulfide-cytochrome-c reductase (flavocytochrome c) 1.8.2.4 dimethylsulfide:cytochrome c2 reductase 1.8.3.1 sulfite oxidase 1.8.7.1 sulfitereductase (ferredoxin) 1.8.98.1 CoB-CoM heterodisulfide reductase1.8.99.1 sulfite reductase 1.8.99.2 adenylyl-sulfate reductase 1.8.99.3hydrogensulfite reductase 1.9.3.1 cytochrome-c oxidase 1.9.6.1 nitratereductase (cytochrome) 1.10.2.2 ubiquinol-cytochrome-c reductase1.10.3.1 catechol oxidase 1.10.3.B1 caldariellaquinol oxidase(H+-transporting) 1.10.3.3 L-ascorbate oxidase 1.10.3.9 photosystem II1.10.3.10 ubiquinol oxidase (H+-transporting) 1.10.3.11 ubiquinoloxidase 1.10.3.12 menaquinol oxidase (H+-transporting) 1.10.9.1plastoquinol-plastocyanin reductase 1.11.1.5 cytochrome-c peroxidase1.11.1.6 Catalase 1.11.1.7 Peroxidase 1.11.1.B2 chloride peroxidase(vanadium-containing) 1.11.1.B7 bromide peroxidase (heme-containing)1.11.1.8 iodide peroxidase 1.11.1.10 chloride peroxidase 1.11.1.11L-ascorbate peroxidase 1.11.1.13 manganese peroxidase 1.11.1.14 ligninperoxidase 1.11.1.16 versatile peroxidase 1.11.1.19 dye decolorizingperoxidase 1.11.1.21 catalase-peroxidase 1.11.2.1 unspecificperoxygenase 1.11.2.2 Myeloperoxidase 1.11.2.3 plant seed peroxygenase1.11.2.4 fatty-acid peroxygenase 1.12.2.1 cytochrome-c3 hydrogenase1.12.5.1 hydrogen:quinone oxidoreductase 1.12.99.6 hydrogenase(acceptor) 1.13.11.9 2,5-dihydroxypyridine 5,6-dioxygenase 1.13.11.11tryptophan 2,3-dioxygenase 1.13.11.49 chlorite O2-lyase 1.13.11.50acetylacetone-cleaving enzyme 1.13.11.52 indoleamine 2,3-dioxygenase1.13.11.60 linoleate 8R-lipoxygenase 1.13.99.3 tryptophan 2′-dioxygenase1.14.11.9 flavanone 3-dioxygenase 1.14.12.17 nitric oxide dioxygenase1.14.13.39 nitric-oxide synthase (NADPH dependent) 1.14.13.17cholesterol 7alpha-monooxygenase 1.14.13.41 tyrosine N-monooxygenase1.14.13.70 sterol 14alpha-demethylase 1.14.13.71 N-methylcoclaurine3′-monooxygenase 1.14.13.81 magnesium-protoporphyrin IX monomethyl ester(oxidative) cyclase 1.14.13.86 2-hydroxyisoflavanone synthase 1.14.13.98cholesterol 24-hydroxylase 1.14.13.119 5-epiaristolochene1,3-dihydroxylase 1.14.13.126 vitamin D3 24-hydroxylase 1.14.13.129beta-carotene 3-hydroxylase 1.14.13.141 cholest-4-en-3-one26-monooxygenase 1.14.13.142 3-ketosteroid 9alpha-monooxygenase1.14.13.151 linalool 8-monooxygenase 1.14.13.156 1,8-cineole2-endo-monooxygenase 1.14.13.159 vitamin D 25-hydroxylase 1.14.14.1unspecific monooxygenase 1.14.15.1 camphor 5-monooxygenase 1.14.15.6cholesterol monooxygenase (side-chain-cleaving) 1.14.15.8 steroid15beta-monooxygenase 1.14.15.9 spheroidene monooxygenase 1.14.18.1Tyrosinase 1.14.19.1 stearoyl-CoA 9-desaturase 1.14.19.3 linoleoyl-CoAdesaturase 1.14.21.7 biflaviolin synthase 1.14.99.1prostaglandin-endoperoxide synthase 1.14.99.3 heme oxygenase 1.14.99.9steroid 17alpha-monooxygenase 1.14.99.10 steroid 21-monooxygenase1.14.99.15 4-methoxybenzoate monooxygenase (O-demethylating) 1.14.99.45carotene epsilon-monooxygenase 1.16.5.1 ascothate ferrireductase(transmembrane) 1.16.9.1 iron:rusticyanin reductase 1.17.1.4 xanthinedehydrogenase 1.17.2.2 lupanine 17-hydroxylase (cytochrome c) 1.17.99.14-methylphenol dehydrogenase (hydroxylating) 1.17.99.2 ethylbenzenehydroxylase 1.97.1.1 chlorate reductase 1.97.1.9 selenate reductase2.7.7.65 diguanylate cyclase 2.7.13.3 histidine kinase 3.1.4.52cyclic-guanylate-specific phosphodiesterase 4.2.1.B9 colneleicacid/etheroleic acid synthase 4.2.1.22 Cystathionine beta-synthase4.2.1.92 hydroperoxide dehydratase 4.2.1.212 colneleate synthase4.3.1.26 chromopyrrolate synthase 4.6.1.2 guanylate cyclase 4.99.1.3sirohydrochlorin cobaltochelatase 4.99.1.5 aliphatic aldoximedehydratase 4.99.1.7 phenylacetaldoxime dehydratase 5.3.99.3prostaglandin-E synthase 5.3.99.4 prostaglandin-I synthase 5.3.99.5Thromboxane-A synthase 5.4.4.5 9,12-octadecadienoate 8-hydroperoxide8R-isomerase 5.4.4.6 9,12-octadecadienoate 8-hydroperoxide 8S-isomerase6.6.1.2 Cobaltochelatase

In some embodiments, the heme protein is an engineered variant or afragment thereof (e.g., a truncated variant containing the heme domain)comprising one or more mutation(s). In some instances, the mutation is asubstitution of the native residue with Ala, Asp, Arg, Asn, Cys, Glu,Gin, Gly, His, lie, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In other embodiments, the heme protein variant is a chimeric proteincomprising recombined sequences or blocks of amino acids from two,three, or more different heme-containing proteins. As described herein,heme protein catalysts described can be improved through theintroduction of mutations which alter the amino acid sequence of theheme protein so as to generate a catalyst that is highly productive andselective for the desired product forming reaction. In particular, thereare many examples in the scientific literature that describe processesthrough which the enantioselectivity and activity of carbene-transferheme proteins can be optimized to form products that do not containcyclopropene moieties or bicyclobutane moieties. Specifically, oneskilled in the art will know that through a process of randommutagenesis via error-prone PCR, or through a process of site-saturationmutagenesis in which one or more codons are randomized sequentially orsimultaneously, or through a process of gene synthesis in which randomor directed mutations are introduced, many different mutants of thegenes encoding the hemoprotein catalysts described herein can begenerated. One skilled in the art will appreciate that heme proteinvariants can be expressed in a whole cell using an expression vectorunder the control of an inducible promoter or by means of chromosomalintegration under the control of a constitutive promotor. The activityof whole cell catalysts, cell lysates or purified proteins can bescreened by GC or HPLC, using parameters including but not limited toturnovers and selectivities as selection criteria to find beneficialmutations.

In some embodiments, the heme enzyme comprises a cytochrome. Cytochromesare a class of heme proteins that are found in bacteria, as well asmitochondria and chloroplasts of eukaryotic organisms, and are typicallyassociated with membranes. Cytochromes typically function in oxidativephosphorylation as components of electron transport chain systems.Cytochromes can be classified by spectroscopy, or by features such asthe structure of the heme group, inhibitor sensitivity, or reductionpotential. Three of the cytochromes, cytochromes a, b, and d, areclassified by their prosthetic group (the prosthetic groups consistingof heme a, heme b, and tetrapyrrolic chelate of iron, respectively).Unlike the aforementioned cytochromes, cytochrome c is not defined interms of its heme group. Cytochrome f, which performs similar functionsto cytochrome c₁ but has a different structure, is sometimes regarded asa type of cytochrome c. Cytochrome P450 proteins form a distinct familyof cytochromes. In bacteria, mitochondria, and chloroplasts, variouscytochromes form different combinations that perform differentfunctions. Cytochromes a and a₃ combine to form cytochrome c oxidase(also known as Complex IV), which is the last enzyme in the respiratorychain of bacteria and mitochondria. Cytochromes b and c₁ combine to formcoenzyme Q—cytochrome c reductase—the third complex in the electrontransport chain. Cytochromes b₆ and f combine to formplastoquinol-plastocyanin reductase, which is found in the chloroplastsof plants, cyanobacteria and green algae and functions inphotosynthesis.

Cytochrome P450 enzymes constitute a large superfamily of heme-thiolateproteins involved in the metabolism of a wide variety of both exogenousand endogenous compounds. Usually, they act as the terminal oxidase inmulticomponent electron transfer chains, such as P450-containingmonooxygenase systems. Members of the cytochrome P450 enzyme familycatalyze myriad oxidative transformations, including, e.g.,hydroxylation, epoxidation, oxidative ring coupling, heteroatom release,and heteroatom oxygenation (E. M. Isin et al., Biochim. Biophys. Acta1770, 314 (2007)). P450s typically contain a single polypeptide, rangingfrom 40 to 55 kDa in molecular weight, and the same general fold hasbeen observed in all P450s with known structures (T. L. Poulous, ChemRev., 114, 3919 (2014)). The active site of these enzymes contains anFe^(III)-protoporphyrin IX cofactor (heme) ligated proximally by aconserved cysteine thiolate (M. T. Green, Current Opinion in ChemicalBiology 13, 84 (2009)). The remaining axial iron coordination site isoccupied by a water molecule in the resting enzyme, but during nativecatalysis, this site is capable of binding molecular oxygen. P450structure is also typically characterized by a long “I helix” (typicallyaround 50 angstroms in length) which runs over the surfaces of the hemeand interacts with oxygen and the oxidation substrate. In the presenceof an electron source, typically provided by NADH or NADPH from anadjacent fused reductase domain or an accessory cytochrome P450reductase enzyme, the heme center of cytochrome P450 activates molecularoxygen, generating a high valent iron(IV)-oxo porphyrin cation radicalspecies intermediate and a molecule of water.

Cytochrome P450 BM3 (CYP102A1) proteins are found in the soil bacteriumBacillus megaterium and catalyze the NADPH-dependent hydroxylation oflong-chain fatty acids at the ω-1 through ω-3 positions. Unlike mostother cytochrome P450 proteins, cytochrome P450 BM3 proteins are anatural fusion between the cytochrome P450 domain and an electrondonating cofactor. Thus, cytochrome P450 BM3 proteins are useful in anumber of biotechnological applications.

In certain embodiments, the hemoprotein is a cytochrome P450 or avariant thereof. In a particular embodiment, the cytochrome P450 is aP450_(BM3) (also known as CYP102A1) or a variant thereof. In someembodiments, the P450_(BM3) hemoprotein is an engineered variantcomprising one or more mutation(s). In some instances, the mutation is asubstitution of the native residue with Ala, Asp, Arg, Asn, Cys, Glu,Gin, Gly, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In other embodiments, the hemoprotein variant is a chimeric proteincomprising recombined sequences or blocks of amino acids from two,three, or more different heme-containing proteins.

In some embodiments, the P450_(BM3) protein (CYP102A1) containsmutations of one or more residues selected from V78, F87, P142, T175,A184, S226, H236, E252, 1263, T268, A290, A328, L353, 1366, C400, T438,and E442 (mature peptide numbering convention) to any other amino acidresidues that is among the naturally occurring twenty amino acids. In afurther embodiment, the P450_(BM3) protein contains a single mutation ofthe residue at position A87 to any other amino acid. In a furtherembodiment, the P450_(BM3) protein contains a single mutation of theresidue at position L181 to any other amino acid. In a furtherembodiment, the P450_(BM3) protein contains a single mutation of theresidue at position F261 to any other amino acid. In a furtherembodiment, the P450_(BM3) protein contains a single mutation of theresidue at position T269 to any other amino acid. In a furtherembodiment, the P450_(BM3) protein contains a single mutation of theresidue at position L437 to any other amino acid. In a furtherembodiment, the P450_(BM3) protein contains a single mutation of theresidue at position V328 to any other amino acid. In a furtherembodiment, the P450_(BM3) protein contains mutations of the residues atpositions F261 and T327 to any other amino acid. In a furtherembodiment, the P450_(BM3) protein contains any combination of mutationsof the residues V78, F87, P142, T175, L181, A184, S226, H236, E252,F261, 1263, T268, T269, A290, T327, A328, L353, 1366, C400, L437, T438,E442 to any other amino acid.

In some embodiments, the heme protein is a cytochrome P450_(BM3) variantincluding the heme domain amino acid sequence set forth in SEQ ID NO:2(P411-P4), and wherein the amino acid sequence optionally comprises oneor more amino acid mutations. In some embodiments, for example, thecytochrome P450_(BM3) variant comprises an amino acid mutation atposition A87 (e.g., an A87F mutation, an A87W mutation, an A87Ymutation, or an A87H mutation). In some embodiments, the A87 mutation isan A87F mutation or an A87W mutation.

In some embodiments, a cytochrome P450_(BM3) variant having the hemedomain amino acid sequence set forth in SEQ ID NO:2 will further containan amino acid mutation at one or both of positions F261 (e.g., F261G)and T327 (e.g., T327P), and optionally an A87F mutation. In someembodiments, the cytochrome P450_(BM3) variant will further contain anamino acid mutation at one to three of positions S72 (e.g., S72W), T269(e.g., T269L), and L437 (e.g., L437F); the variant may optionallycontain an A87F mutation in such cases.

The cytochrome P450_(BM3) variant having the heme domain amino acidsequence set forth in SEQ ID NO:2 can also contain an amino acidmutation at one or both of positions A78 (e.g., A78F) and A330 (e.g.,A330V), and optionally an A87F mutation. Such variants may also containany of the mutations at positions S72, F261, T269, T327, and L437described above.

In some embodiments, the cytochrome P450_(BM3) variant comprisesmutations at one or both of positions L188 (e.g., L188C) and T436 (e.g.,T436M), and optionally an A87F mutation. Such variants may also containany of the mutations at positions S72, A78, F261, T269, T327, A330, andL437 described above.

In some embodiments, a cytochrome P450_(BM3) variant having the hemedomain amino acid sequence set forth in SEQ ID NO:2 will further containan amino acid mutation at position L437 (e.g., L437G), and optionally anA87W mutation. In some embodiments, the cytochrome P450_(BM3) variantwill further contain an amino acid mutation at one or both of positionsT327 (e.g., T327V) and V328 (e.g., V328Y); the variant may optionallycontain an A87W mutation in such cases.

The cytochrome P450_(BM3) variant having the heme domain amino acidsequence set forth in SEQ ID NO:2 can also contain an amino acidmutation at one or both of positions F261 (e.g., F261M) and T436 (e.g.,T436H), and optionally an A87W mutation. Such variants may also containany of the mutations at positions T327, V328, and L437 described above.

P450_(BM3) variants having a heme domain amino acid sequence accordingto SEQ ID NO:2, including those having the mutations set forth above,can be particularly useful for forming cyclopropene products.Full-length P450_(BM3) variants comprising the amino acid sequenceaccording to SEQ ID NO:5, and any mutations corresponding to thosedescribed above, can also be used in the methods provided herein. Theheme-domain polypeptide sequences (e.g., a heme domain amino acidsequence according to SEQ ID NO:2) and the full-length polypeptidesequences (e.g., a full-length amino acid sequence according to SEQ IDNO:5) may further contain an N-terminal methionine residue directlyproceeding the T1 position in certain instances.

In some embodiments, the heme protein is a cytochrome P450_(BM3) variantcomprising the heme domain amino acid sequence set forth in SEQ ID NO:3(P411-E10), and which optionally comprises one or more amino acidmutations. For example, the variant can contain a mutation a positionV78 in SEQ ID NO:3 (e.g., a V78F mutation, a V78Y mutation, a V78Hmutation, or a V78W mutation). In some embodiments, the V78 mutation isa V78F mutation or a V78Y mutation. In some embodiments, the cytochromeP450_(BM3) variant comprises an amino acid mutation at position S438(e.g., an S438 mutation).

P450_(BM3) variants having a heme domain amino acid sequence accordingto SEQ ID NO:3, including those having the mutations set forth above,can be particularly useful for forming bicyclobutane products.Full-length P450_(BM3) variants comprising the amino acid sequenceaccording to SEQ ID NO:6, and any mutations corresponding to thosedescribed above, can also be used in the methods provided herein. Theheme-domain polypeptide sequences (e.g., a heme domain amino acidsequence according to SEQ ID NO:3) and the full-length polypeptidesequences (e.g., a full-length amino acid sequence according to SEQ IDNO:6) may further contain an N-terminal methionine residue directlyproceeding the T1 position in certain instances.

Other cytochrome P450 enzymes can be used in the methods for formingcyclopropenes and bicyclobutanes. For example, the P450 may be P. putidaP450cam having the amino acid structure set forth in SEQ ID NO:7 or avariant thereof having one or more mutations. For example, a P450cam maycontain one or more mutations at positions P89, Y96, F98, T101, V247,D251, N255, L294, V295, A296, D297, C357, 1395, and V396 in SEQ ID NO:7.Cytochrome P450 variants with sequences similar to SEQ ID NOS: 4-7(e.g., sequences with at least 70%, 75%, 80%, 85%, 90%, or 95% sequenceidentity to SEQ ID NOS: 4-7), may also contain mutations at thepositions corresponding to P89, Y96, F98, T101, V247, D251, N255, L294,V295, A296, D297, C357, 1395, and V396 of SEQ ID NO:7.

In some embodiments, the heme enzyme comprises a globin enzyme. Globinsare a superfamily of globular heme proteins that are typically involvedin the transport and binding of oxygen. A characteristic of globins is athree-dimensional fold consisting of eight alpha helices, often labeledA-H, that can fold into a three-over-three sandwich structure. Someglobins also additional terminal helix extensions. So-called “truncatedhemoglobins” contain four alpha helices arranged in a two-over-twosandwich. Globins can be divided into three groups: single-domainglobins, flavohemoglobins (not observed in archaea), and globin-coupledsensors (not observed in eukaryotes). All three groups are observed inbacteria. Globin proteins include hemoglobin, myoglobin, neuroglobin,cytoglobin, erythrocruorin, leghemoglobin, non-symbiotic hemoglobin,flavohemoglobins (one group of chimeric globins), globin E,globin-coupled sensors (another group of chimeric globins), protoglobin,truncated 2/2 globin, HbN, cyanoglobin, HbO, and Glb3.

In some embodiments, the heme protein used for formation of cyclopropeneand/or bicyclobutane products is a globin or a variant thereof. Forexample, the globin may be sperm whale myoglobin comprising the aminoacid sequence set forth in SEQ ID NO:8, or a variant thereof containingone or more mutations. In some embodiments, a sperm whale myoglobinvariant may contain a mutation at H64 (e.g., an H64V mutation) or V68(e.g., a V68A mutation). In some embodiments, the myoglobin variant mayfurther contain mutations at L29 and/or F43. Such mutations may also bemade at the corresponding positions of other globins such thosecontaining a three-over-three helix sandwich fold (including but notlimited to, C. jejuni globin (SEQ ID NO:9), V. stercoraria hemoglobin(SEQ ID NO:10), murine neuroglobin (SEQ ID NO:11), human neuroglobin(SEQ ID NO:12), an M. infernorum hemoglobin (SEQ ID NO:13), humancytoglobin (SEQ ID NO:14), and A. suum hemoglobin (SEQ ID NO:15).

In some embodiments, the globin is a truncated globin such as B.subtilis truncated hemoglobin comprising the amino sequence set forth inSEQ ID NO:16 or a variant thereof having one or more mutations. One ormore mutations may reside within the distal binding pocket of B.subtilis truncated hemoglobin, for example at T45 and/or at Q49 withrespect to SEQ ID NO:16, or within the analogous regions of otherglobins such as those containing a two-over-two helix sandwich fold.

Protoglobins were the first globins identified in Archaea such as M.acetivorans, A. pernix, and P. ferrireducens. Protoglobin tertiarystructure frequently includes the canonical globin fold, as well as apre-A helix (termed “Z” in certain instances) and an N-terminalextension. In some embodiments, the heme protein used for formation ofcyclopropene and/or bicyclobutane products is a protoglobin or a variantthereof. For example, the protoglobin may be an M. acetivoransprotoglobin comprising the amino acid sequence set forth in SEQ IDNO:17, or a variant thereof containing one or more mutations; an A.pernix protoglobin comprising the amino acid sequence set forth in SEQID NO:18, or a variant thereof containing one or more mutations; or a P.ferrireducens protoglobin comprising the amino acid sequence set forthin SEQ ID NO:19, or a variant thereof containing one or more mutations.For example the protoglobin variant may contain one or mutations atpositions W59, Y60, F73, F145, or F93 in SEQ ID NO:18, or at analogouspositions in other protoglobins (e.g., protoglobins with at least 70%,75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO:18).

One skilled in the art will appreciate that the P450_(BM3) catalysts andother enzyme catalysts described herein can be improved through theintroduction of additional DNA mutations which alter the resulting aminoacid sequence of the hemoprotein so as to generate a catalyst that ishighly productive and selective for the desired cyclopropenation andbicyclobutanation reactions. In particular, there are many examples inthe scientific literature that describe processes through which theenantioselectivity and activity of carbene-transfer hemoproteins can beoptimized to produce products that do not include cyclopropenes andbicyclobutanes. Specifically, one skilled in the art will know thatthrough a process of random mutagenesis via error-prone PCR, or througha process of site-directed mutagenesis in which one or more codons arerandomized sequentially or simultaneously, or through a process of genesynthesis in which random or directed mutations are introduced, manydifferent mutants of the genes encoding the P450_(BM3) enzymes and otherenzymes described herein can be generated. One skilled in the art willappreciate that heme protein variants can be expressed in a whole cellusing an expression vector under the control of an inducible promoter orby means of chromosomal integration under the control of a constitutivepromoter. The cyclopropenation and bicyclobutanation activities of wholecell catalysts, or purified proteins can be screened by GC or HPLC,using parameters including but not limited to turnovers andselectivities as selection criteria to find beneficial mutations.

One skilled in the art will understand that P450_(BM3) mutants and otherenzymes identified as improved in the cyclopropenation andbicyclobutanation can themselves be subjected to additional mutagenesisas described herein, resulting in progressive, cumulative improvementsin one or more reaction parameters including but not limited to turnoverfrequency, total turnover number, yield, chemoselectivity,regioselectivity, diastereoselectivity, enantioselectivity, expression,thermostability, or solvent tolerance.

In some embodiments, the heme protein, homolog, variant, or fragmentthereof has a turnover frequency (TOF) between about 1 min⁻¹ and 10min⁻¹ (e.g., about 1 min⁻¹, 1.5 min⁻¹, 2 min⁻¹, 2.5 min⁻¹, 3 min⁻¹, 3.5min⁻¹, 4 min⁻¹, 4.5 min⁻¹, 5 min⁻¹, 5.5 min⁻¹, 6 min⁻¹, 6.5 min⁻¹, 7min⁻¹, 7.5 min⁻¹, 8 min⁻¹, 8.5 min⁻¹, 9 min⁻¹, 9.5 min⁻¹, or 10 min⁻¹).In other embodiments, the TOF is between about 10 min⁻¹ and 100 min⁻¹(e.g., about 10 min⁻¹, 11 min⁻¹, 12 min⁻¹, 13 min⁻¹, 14 min⁻¹, 15 min⁻¹,16 min⁻¹, 17 min⁻¹, 18 min⁻¹, 19 min⁻¹, 20 min⁻¹, 21 min⁻¹, 22 min⁻¹, 23min⁻¹, 24 min⁻¹, 25 min⁻¹, 26 min⁻¹, 27 min⁻¹, 28 min⁻¹, 29 min⁻¹, 30min⁻¹, 31 min⁻¹, 32 min⁻¹, 33 min⁻¹, 34 min⁻¹, 35 min⁻¹, 36 min⁻¹, 37min⁻¹, 38 min⁻¹, 39 min⁻¹, 40 min⁻¹, 41 min⁻¹, 42 min⁻¹, 43 min⁻¹, 44min⁻¹, 45 min⁻¹, 46 min⁻¹, 47 min⁻¹, 48 min⁻¹, 49 min⁻¹, 50 min⁻¹, 55min⁻¹, 60 min⁻¹, 65 min⁻¹, 70 min⁻¹, 75 min⁻¹, 80 min⁻¹, 85 min⁻¹, 90min⁻¹, 95 min⁻¹, or 100 min⁻¹). In other instances, the TOF is greaterthan about 100 min⁻¹ to 1,000 min⁻¹ (e.g., greater than about 100 min⁻¹,150 min⁻¹, 200 min⁻¹, 250 min⁻¹, 300 min⁻¹, 350 min⁻¹, 400 min⁻¹, 450min⁻¹, 500 min⁻¹, 550 min⁻¹, 600 min⁻¹, 650 min⁻¹, 700 min⁻¹, 750 min⁻¹,800 min⁻¹, 850 min⁻¹, 900 min⁻¹, 950 min⁻¹, 1,000 min⁻¹, or more). Insome instances, the TOF is greater than about 10 min⁻¹. In otherinstances, the TOF is greater than about 45 min⁻¹.

In other embodiments, the heme protein, homolog, variant, or fragmentthereof has a total turnover number (TTN), which refers to the maximumnumber of molecules of a substrate that the protein can convert beforebecoming inactivated, of between about 1 and 100 (e.g., about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100). In someother embodiments, the TTN is between about 100 and 1,000 (e.g., about100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, or 1,000). In some embodiments, the TTN is betweenabout 1,000 and 2,000 (e.g., about 1,000, 1,050, 1,100, 1,150, 1,200,1,250, 1,300, 1,350, 1,400, 1,450, 1,500, 1,550, 1,600, 1,650, 1,700,1,750, 1,800, 1,850, 1,900, 1,950 or 2,000). In other embodiments, theTTN is at least about 2,000 (e.g., at least about 2,000, 2,500, 3,000,3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000,8,500, 9,000, 9,500, or 10,000). In some instances, the TTN is greaterthan about 70. In other instances, the TTN is greater than about 1,800.

In some embodiments, the heme protein variant or fragment thereof hasenhanced activity of at least about 1.5 to 2,000 fold (e.g., at leastabout 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,050,1,100, 1,150, 1,200, 1,250, 1,300, 1,350, 1,400, 1,450, 1,500, 1,550,1,600, 1,650, 1,700, 1,750, 1,800, 1,850, 1,900, 1,950, 2,000, or more)fold compared to the corresponding wild-type heme protein.

In some embodiments, activity is expressed in terms of turnoverfrequency (TOF). In particular embodiments, the TOF of the heme proteinvariant or fragment thereof is at least about 1.5, 2, 2.5, 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 fold higher than thecorresponding wild-type protein.

In other instances, activity is expressed in terms of total turnovernumber (TTN). In particular instances, the TTN of the theme proteinvariant or fragment thereof is about least about 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000,1,050, 1,100, 1,150, 1,200, 1,250, 1,300, 1,350, 1,400, 1,450, 1,500,1,550, 1,600, 1,650, 1,700, 1,750, 1,800, 1,850, 1,900, 1,950, or 2,000fold higher than the corresponding wild-type protein.

In some embodiments, the present invention provides heme proteins,homologs, variants, and fragments thereof that catalyze enantioselectivecarbene insertion into alkyne carbon-carbon bonds with high enantiomericexcess. In particular embodiments, the heme proteins are variants orfragments thereof that catalyze enantioselective carbene insertion intoalkyne bonds with higher enantiomeric excess values than that of thecorresponding wild-type protein. In some embodiments, the heme protein,homolog, variants, or fragment thereof catalyzes carbene insertion intoalkyne bonds with an enantiomeric excess value of at least about 30% ee(e.g., at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% ee). Preferably, the heme protein, homolog,variant, or fragment thereof catalyzes carbene insertion into alkynebonds with at least about 80% ee (e.g., at least about 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% ee). More preferably, the heme protein, homolog,variant, or fragment thereof catalyzes carbene insertion into alkynebonds with at least about 95% ee (e.g., at least about 95%, 96%, 97%,98%, 99%, or 100% ee).

In certain embodiments, a conserved residue in a heme protein ofinterest that serves as an heme axial ligand can be identified bylocating the segment of the DNA sequence in the corresponding gene whichencodes the conserved residue. In some instances, this DNA segment isidentified through detailed mutagenesis studies in a conserved region ofthe protein. In other instances, the conserved residue is identifiedthrough crystallographic study.

In situations where detailed mutagenesis studies and crystallographicdata are not available for a heme protein of interest, the axial ligandmay be identified through phylogenetic study. Due to the similarities inamino acid sequence within families of heme proteins (e.g., cytochrome cproteins), standard protein alignment algorithms may show a phylogeneticsimilarity between a heme protein for which crystallographic ormutagenesis data exist and a new heme protein for which such data do notexist. Thus, the polypeptide sequences of the present invention forwhich the heme axial ligand is known can be used as a “query sequence”to perform a search against a specific new heme protein of interest or adatabase comprising heme protein sequences to identify the heme axialligand. Such analyses can be performed using the BLAST programs (see,e.g., Altschul et al., J Mol Biol. 215(3):403-10(1990)). Software forperforming BLAST analyses publicly available through the National Centerfor Biotechnology Information (http://ncbi.nlm.nih.gov). BLASTP is usedfor amino acid sequences.

Exemplary parameters for performing amino acid sequence alignments toidentify the heme axial ligand in a heme protein of interest using theBLASTP algorithm include E value=10, word size=3, Matrix=Blosum62, Gapopening=11, gap extension=1, and conditional compositional score matrixadjustment. Those skilled in the art will know what modifications can bemade to the above parameters, e.g., to either increase or decrease thestringency of the comparison and/or to determine the relatedness of twoor more sequences.

In some embodiments, the heme protein comprises an amino acid sequencethat has about 70% or greater (e.g., about 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity toany of the amino acid sequences described herein (e.g., any of the aminoacid sequences set forth in SEQ ID NOS:1-6). In other embodiments, theheme protein comprises an amino acid sequence that has about 80% orgreater (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity toany of the amino acid sequences described herein. In particularembodiments, the heme protein comprises an amino acid sequence that hasabout 90% or greater (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identity any of the amino acid sequencesdescribed herein. In some instances, the heme protein comprises an aminoacid sequence that is about 95%, 96,%, 97%, 98%, 99%, or 100% identicalany of the amino acid sequences described herein.

In some embodiments, the heme protein comprises an amino acid sequencethat contains between about 5 and 124 (e.g., about 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, or 124) of theamino acids in SEQ ID NOS:1-19, or variants thereof as described above.The amino acids may be contiguous, or separated by any number of aminoacids.

In certain embodiments, mutations can be introduced into the target geneusing standard cloning techniques (e.g., site-directed mutagenesis,site-saturated mutagenesis) or by gene synthesis to produce the hemeproteins, fragments thereof, variants thereof, or homologs thereof ofthe present invention. In some embodiments, the heme protein, fragmentthereof, variant thereof, or homolog thereof is recombinantly expressedand optionally isolated and/or purified for carrying out the in vitrocarbene insertion reactions of the present invention. In otherembodiments, the heme protein, fragment thereof, variant thereof, orhomolog thereof is expressed in whole cells such as bacterial cells,archaeal cells, yeast cells, fungal cells, insect cells, plant cells, ormammalian cells, and these cells are used for carrying out the in vivocarbene insertion reactions of the present invention. The wild-type ormutated gene can be expressed in a whole cell using an expression vectorunder the control of an inducible promoter or by means of chromosomalintegration under the control of a constitutive promoter. Carbeneinsertion activity can be screened in vivo or in vitro by followingproduct formation by GC or HPLC. Suitable bacterial host cells include,but are not limited to, BL21 E. coli, DE3 strain E. coli, E. coli M15,DH5a, DH10f3, HB101, T7 Express Competent E. coli (NEB), B. subtiliscells, Pseudomonas fluorescens cells, and cyanobacterial cells such asChlamydomonas reinhardtii cells and Synechococcus elongates cells.Non-limiting examples of archaeal host cells include Pyrococcusfuriosus, Metallosphera sedula, Thermococcus litoralis, Methanobacteriumthermoautotrophicum, Methanococcus jannaschii, Pyrococcus abyssi,Sulfolobus solfataricus, Pyrococcus woesei, Sulfolobus shibatae, andvariants thereof. Fungal host cells include, but are not limited to,yeast cells from the genera Saccharomyces (e.g., S. cerevisiae), Pichia(P. Pastoris), Kluyveromyces (e.g., K. lactis), Hansenula and Yarrowia,and filamentous fungal cells from the genera Aspergillus, Trichoderma,and Myceliophthora. Suitable insect host cells include, but are notlimited to, Sf9 cells from Spodoptera frugiperda, Sf21 cells fromSpodoptera frugiperda, Hi-Five cells, BTI-TN-5B1-4 Trichophusia m cells,and Schneider 2 (S2) cells and Schneider 3 (S3) cells from Drosophilamelanogaster. Non-limiting examples of mammalian host cells includeHEK293 cells, HeLa cells, CHO cells, COS cells, Jurkat cells, NSOhybridoma cells, baby hamster kidney (BHK) cells, MDCK cells, NIH-3T3fibroblast cells, and any other immortalized cell line derived from amammalian cell. Non-limiting examples of plant host cells include thosefrom tobacco, tomato, potato, maize, rice, lettuce, and spinach. Ingeneral, cells from plants that have short generation times and/or yieldreasonable biomass with standard cultivation techniques are preferable.

In certain embodiments, the present invention provides the hemeproteins, fragments thereof, variants thereof, or homologs thereof, suchas the cytochrome c variants described herein that are active carbeneinsertion catalysts, inside living cells. As a non-limiting example,bacterial cells (e.g., E. coli) can be used as host whole cell catalystsfor the in vivo carbene insertion reactions of the present invention,although any number of host whole cells may be used, including but notlimited to the host cells described herein. In some embodiments, hostwhole cell catalysts containing heme proteins, fragments thereof,variants thereof, or homologs thereof are found to significantly enhancethe total turnover number (TTN) compared to the in vitro reactions usingisolated heme proteins, fragments thereof, variants thereof, or homologsthereof.

The expression vector comprising a nucleic acid sequence that encodes aheme protein, fragment thereof, variant thereof, or homolog thereof ofthe invention can be a viral vector, a plasmid, a phage, a phagemid, acosmid, a fosmid, a bacteriophage (e.g., a bacteriophage P1-derivedvector (PAC)), a baculovirus vector, a yeast plasmid, or an artificialchromosome (e.g., bacterial artificial chromosome (BAC), a yeastartificial chromosome (YAC), a mammalian artificial chromosome (MAC),and human artificial chromosome (HAC)). Expression vectors can includechromosomal, non-chromosomal, and synthetic DNA sequences. Equivalentexpression vectors to those described herein are known in the art andwill be apparent to the ordinarily skilled artisan.

The expression vector can include a nucleic acid sequence encoding aheme protein, fragment thereof, variant thereof, or homolog thereof thatis operably linked to a promoter, wherein the promoter comprises aviral, bacterial, archaeal, fungal, insect, plant, or mammalianpromoter. In certain embodiments, the promoter is a constitutivepromoter. In some embodiments, the promoter is an inducible promoter. Inother embodiments, the promoter is a tissue-specific promoter or anenvironmentally regulated or a developmentally regulated promoter.

In some embodiments, the nucleic acid sequence encodes a heme proteinthat comprises an amino acid sequence that has about 70% or greater(e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence setforth in SEQ ID NOS:1-19. In other embodiments, the nucleic acidsequence encodes a heme protein that comprises an amino acid sequencethat has about 80% or greater (e.g., about 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identity to the amino acid sequence set forth in SEQ ID NOS:1-19.In particular embodiments, the nucleic acid sequence encodes a hemeprotein that comprises an amino acid sequence that has about 90% orgreater (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identity to the amino acid sequence set forth in SEQ IDNOS:1-19. In some instances, the nucleic acid sequence encodes a hemeprotein that comprises an amino acid sequence that is about 95%, 96,%,97%, 98%, 99%, or 100% identical to the amino acid sequence set forth inSEQ ID NOS:1-19.

In other embodiments, the nucleic acid sequence encodes a heme proteinthat comprises an amino acid sequence that contains between about 5 and124 (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, or 124) of the amino acids in SEQ ID NOS:1-19.The amino acids may be contiguous, or separated by any number of aminoacids.

It is understood that affinity tags may be added to the N- and/orC-terminus of a heme protein, fragment thereof, variant thereof, orhomolog thereof expressed using an expression vector to facilitateprotein purification. Non-limiting examples of affinity tags includemetal binding tags such as His6-tags and other tags such as glutathioneS-transferase (GST).

Non-limiting expression vectors for use in bacterial host cells includepCWori, pET vectors such as pET22 (EMD Millipore), pBR322 (ATCC₃₇₀₁₇),pQE™ vectors (Qiagen), pBluescript™ vectors (Stratagene), pNH vectors,lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T(Pharmacia), pRSET, pCR-TOPO vectors, pET vectors, pSyn_1 vectors,pChlamy_1 vectors (Life Technologies, Carlsbad, Calif.), pGEM1 (Promega,Madison, Wis.), and pMAL (New England Biolabs, Ipswich, Mass.).Non-limiting examples of expression vectors for use in eukaryotic hostcells include pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40(Pharmacia), pcDNA3.3, pcDNA4/TO, pcDNA6/TR, pLenti6/TR, pMT vectors(Life Technologies), pKLAC1 vectors, pKLAC2 vectors (New EnglandBiolabs), pQE™ vectors (Qiagen), BacPak baculoviral vectors, pAdeno-X™adenoviral vectors (Clontech), and pBABE retroviral vectors. Any othervector may be used as long as it is replicable and viable in the hostcell.

B. Cyclopropene Products

In some embodiments, methods for producing cyclopropene productscomprise combining an alkyne, a carbene precursor, and a heme protein,homolog thereof, variant thereof, or fragment thereof as describedherein under conditions sufficient to form the cyclopropene.

In some embodiments, the alkyne is a compound according to Formula I:

wherein

R¹⁰ is selected from the group consisting of H and CR¹R²; each R¹ isindependently selected from the group consisting of H, C₁₋₁₈ alkyl,C₁₋₁₈ haloalkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, (C₃₋₁₀ (C₆₋₁₀ aryl)-L¹-,(5- to 10-membered heteroaryl)-L¹-, (5- to 10-memberedheterocyclyl)-L¹-, cyano, halo, nitro, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷,C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂, C(O)NR⁷OR⁸, C(O)C(O)OR⁷, andP(O)(OR⁷)₂;

C₁₋₁₈ alkyl, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, 5- to 10-membered heteroaryl,and 5- to 10-membered heterocyclyl in R¹ are optionally andindependently substituted with one or more R^(1a);

each R^(1a) is independently selected from the group consisting ofhalogen, cyano, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ heteroalkyl, C₂₋₆alkynyl, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, and 5- to 10-memberedheterocyclyl;

L¹ is selected from the group consisting of a bond and C₁₋₂₀ alkylene;

when L¹ is C₂₋₂₀ alkylene, one or more non-adjacent CH₂ groups areoptionally and independently replaced with O, S, or NH;

when L¹ is C₃₋₂₀ alkylene, one or more pairs of adjacent CH₂ groups areoptionally and independently replaced with C(O)O or C(O)NH;

each R² is independently selected from the group consisting of H, C₁₋₁₈alkyl, C₁₋₁₈ haloalkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, (C₃₋₁₀cycloalkyl)-L²-, (C₆₋₁₀ aryl)-L²-, (5- to 10-membered heteroaryl) L²-,(5- to 10-membered heterocyclyl)-L²-, cyano, halo, nitro, N(R⁸)₂,B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷, C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂,C(O)NR⁷ORz⁸, C(O)C(O)OR⁷, and P(O)(OR⁷)₂;

C₁₋₁₈ alkyl, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, 5- to 10-membered heteroaryl,and 5- to 10-membered heterocyclyl in R² are optionally andindependently substituted with one or more R^(2a);

each R^(2a) is independently selected from the group consisting ofhalogen, cyano, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ heteroalkyl, C₂₋₆alkynyl, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, and 5- to 10-memberedheterocyclyl;

L² is selected from the group consisting of a bond and C₁₋₂₀ alkylene;

when L² is C₂₋₂₀ alkylene, one or more non-adjacent CH₂ groups areoptionally and independently replaced with O, S, or NH;

when L² is C₃₋₂₀ alkylene, one or more pairs of adjacent CH₂ groups areoptionally and independently replaced with C(O)O or C(O)NH; and

each R⁷, R⁸, and R⁹ is independently selected from the group consistingof H, optionally substituted C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl,optionally substituted C₆₋₁₀ aryl, optionally substituted 5- to10-membered heteroaryl, and optionally substituted 5- to 10-memberedheterocyclyl.

In some embodiments, the alkyne is a compound according to Formula Ia:

In some embodiments, each R² in alkynes of Formula I is independentlyselected from H and C₁₋₆ alkyl. In some embodiments, R² in alkynes ofFormula Ia is selected from H and C₁₋₆ alkyl. R² can be, for example, H,methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, isopentyl, or n-hexyl. In some embodiments, R² inalkynes according to Formula I or Formula Ia is H.

In some embodiments, R¹ is selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkynyl, (C₃₋₁₀ cycloalkyl)-L¹-, (C₆₋₁₀ aryl)-L¹-, (5- to10-membered heteroaryl)-L¹-, and (5- to 10-membered heterocyclyl)-L¹-,each of which is optionally and independently substituted with one ormore R^(1a). In some embodiments, R¹ is selected from the groupconsisting of C₁8 alkyl and C₂₋₈ alkynyl, each of which is optionallysubstituted with one or more R^(1a). R¹ can be, for example, optionallysubstituted methyl, optionally substituted ethyl, optionally substitutedn-propyl, optionally substituted isopropyl, optionally substitutedn-butyl, optionally substituted isobutyl, optionally substitutedsec-butyl, optionally substituted tert-butyl, optionally substitutedn-pentyl, optionally substituted isopentyl, optionally substitutedn-hexyl, optionally substituted branched hexyl, optionally substitutedn-heptyl, optionally substituted branched heptyl, optionally substitutedn-octyl, optionally substituted branched octyl, optionally substitutedethynyl, optionally substituted prop-1-yn-1-yl, optionally substitutedprop-2-yn-1-yl, optionally substituted but-1-yn-1-yl, optionallysubstituted but-2-yn-1-yl, optionally substituted but-3-yn-1-yl,optionally substituted pent-1-yn-1-yl, optionally substitutedpent-2-yn-1-yl, optionally substituted pent-3-yn-1-yl, optionallysubstituted pent-4-yn-1-yl, optionally substituted hex-1-yn-1-yl,optionally substituted hex-2-yn-1-yl, optionally substitutedhex-3-yn-1-yl, optionally substituted hex-4-yn-1-yl, optionallysubstituted hex-5-yn-1-yl, optionally substituted hept-1-yn-1-yl,optionally substituted hept-2-yn-1-yl, optionally substitutedhept-3-yn-1-yl, optionally substituted hept-4-yn-1-yl, optionallysubstituted hept-5-yn-1-yl, optionally substituted hept-6-yn-1-yl,optionally substituted oct-1-yn-1-yl, optionally substitutedoct-2-yn-1-yl, optionally substituted oct-3-yn-1-yl, optionallysubstituted oct-4-yn-1-yl, optionally substituted oct-5-yn-1-yl,optionally substituted oct-6-yn-1-yl, or optionally substitutedoct-7-yn-1-yl. Alkynes according to Formula I or Formula Ia havingalkenyl R¹ groups, cycloalkyl R¹ _(g)roups, and the like can also beemployed.

In some embodiments, R¹ in alkynes according to Formula I is selectedfrom the group consisting of C₁₋₆ alkyl and C₂₋₆ alkynyl, each of whichis optionally substituted with one or more R^(1a), and R¹⁰ is H. In somesuch embodiments, R¹ is C₁₋₆ alkyl and R^(1a) is selected from halogen(e.g., Cl or Br), —OH, C₁₋₆ alkoxy (e.g., methoxy, ethoxy, n-propoxy, orisopropoxy), and C₁₋₆ heteroalkyl (e.g., methoxymethyl or ethoxymethyl).

In some embodiments, R¹ is selected from (C₃₋₁₀ cycloalkyl)-L¹-, (C₆₋₁₀aryl)-L¹, (5- to 10-membered heteroaryl)-L¹-, and (5- to 10-memberedheterocyclyl)-L¹-, wherein cycloalkyl, aryl, heteroaryl, andheterocyclyl are optionally substituted with one or more R^(1a). In someembodiments, L¹ is a covalent bond. For example, the grouping (C₃₋₁₀cycloalkyl)-L¹- can be optionally substituted cyclopropyl, optionallysubstituted cyclobutyl, optionally substituted cyclopentyl, optionallysubstituted cyclohexyl, or the like. In some embodiments, L¹ is C₁₋₂₀alkylene (e.g., C₁₋₁₀ alkylene or C₁₋₆ alkylene). For example, thegrouping (C₃₋₁₀ cycloalkyl)-L¹- can be cyclohexylmethyl,(2-cyclohexyl)eth-1-yl, (3-cyclohexyl)prop-1-yl, or the like. Thegrouping (C₆₋₁₀ aryl)-L¹- can be, for example, phenethyl. Cycloalkylgroups, aryl groups, and heterocyclyl groups can be further substitutedwith R^(1a) groups as described above. In some embodiments, R¹ iscyclopropyl, cyclobutyl, cyclohexyl, 1,3-dioxolan-2-yl, ortetrahydropyran-1-yl; and R^(1a) is —OH, C₁₋₆ alkoxy (e.g., methoxy,ethoxy, n-propoxy, or isopropoxy), or C₁₋₆ heteroalkyl (e.g.,methoxymethyl or ethoxymethyl).

In some embodiments, L¹ is C₁₋₂₀ alkylene where one or more non-adjacentCH₂ groups are optionally and independently replaced with O, S, or NH.For example, the L¹ moiety may have the formula—(CH₂)_(w)(L^(1a))_(x)(CH₂)_(y)(L^(1a))_(x)(CH₂)_(z)—, where each L^(1a)is independently O, S, or NH; each subscript x is independently 0 or 1;and each of subscripts w, y, and z is a non-zero integer such that thesum of subscript w, subscript x, subscript y, and subscript z is lessthan or equal to 20. In some such embodiments, each L^(1a) is 0. In someembodiments, the L¹ moiety is —CH₂O(CH₂)₆—.

In some embodiments, L¹ is C₃₋₂₀ alkylene wherein one or more pairs ofadjacent CH₂ groups are optionally and independently replaced with C(O)Oor C(O)NH. For example, the L¹moiety may have the formula—(CH₂)_(q)(L^(1b))_(r)(CH₂)_(s)(L^(1b))_(t)(CH₂)_(u)—, where each L^(1b)is independently C(O)O or C(O)NH; subscript r and subscript t areindependently 0 or 1; and each of subscripts q, s, and u is a non-zerointeger such that: 1) the sum of subscript q, subscript s, and subscriptu is less than or equal to 20 when subscript r and subscript t are 0; 2)the sum of subscript q, subscript s, and subscript u is less than orequal to 18 when the sum of subscript r and subscript t is 1; or 3) thesum of subscript q, subscript s, and subscript u is less than or equalto 16 when the sum of subscript r and subscript t is 2. In someembodiments, the L¹ moiety is —C(O)O(CH₂)₆—. L¹ groups having both oneor more C^(1a) moieties (i.e., O, S, or NH) and one or more L^(1b)moieties (i.e., C(O)O or C(O)NH) can also be employed.

In some embodiments, the alkyne for forming cyclopropene products isselected from:

In some embodiments, the alkyne for forming cyclopropene products isselected from:

In some embodiments, the alkyne for forming cyclopropene products isselected from:

A number of carbene precursors can be used in the methods and reactionmixtures of the invention including, but not limited to, amines, azides,hydrazines, hydrazones, epoxides, diazirenes, and diazo reagents. Insome embodiments, the carbene precursor is an epoxide (i.e., a compoundcontaining an epoxide moiety). The term “epoxide moiety” refers to athree-membered heterocycle having two carbon atoms and one oxygen atomconnected by single bonds. In some embodiments, the carbene precursor isa diazirene (i.e., a compound containing a diazirine moiety). The term“diazirine moiety” refers to a three-membered heterocycle having onecarbon atom and two nitrogen atoms, wherein the nitrogen atoms areconnected via a double bond. Diazirenes are chemically inert, smallhydrophobic carbene precursors described, for example, in US2009/0211893, by Turro (J. Am. Chem. Soc. 1987, 109, 2101-2107), and byBrunner (J. Biol. Chem. 1980, 255, 3313-3318), which are incorporatedherein by reference in their entirety.

In some embodiments, the carbene precursor is a diazo reagent, e.g., anα-diazoester, an α-diazoamide, an α-diazonitrile, an α-diazoketone, anα-diazoaldehyde, and an α-diazosilane. Diazo reagents can be formed froma number of starting materials using procedures that are known to thoseof skill in the art. Ketones (including 1,3-diketones), esters(including (β-ketones), acyl chlorides, and carboxylic acids can beconverted to diazo reagents employing diazo transfer conditions with asuitable transfer reagent (e.g., aromatic and aliphatic sulfonyl azides,such as toluenesulfonyl azide, 4-carboxyphenylsulfonyl azide,2-naphthalenesulfonyl azide, methylsulfonyl azide, and the like) and asuitable base (e.g., triethylamine, triisopropylamine,diazobicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]undec-7-ene, and thelike) as described, for example, in U.S. Pat. No. 5,191,069 and byDavies (J. Am. Chem. Soc. 1993, 115, 9468-9479), which are incorporatedherein by reference in their entirety. The preparation of diazocompounds from azide and hydrazone precursors is described, for example,in U.S. Pat. Nos. 8,350,014 and 8,530,212, which are incorporated hereinby reference in their entirety. Alkylnitrite reagents (e.g.,(3-methylbutyl)nitrite) can be used to convert α-aminoesters to thecorresponding diazo compounds in non-aqueous media as described, forexample, by Takamura (Tetrahedron, 1975, 31: 227), which is incorporatedherein by reference in its entirety. Alternatively, a diazo compound canbe formed from an aliphatic amine, an aniline or other arylamine, or ahydrazine using a nitrosating agent (e.g., sodium nitrite) and an acid(e.g., p-toluenesulfonic acid) as described, for example, by Zollinger(Diazo Chemistry I and II, VCH Weinheim, 1994) and in US 2005/0266579,which are incorporated herein by reference in their entirety.

In some embodiments, the carbene precursor is a diazo compound such as acompound according to Formula II:

wherein

R³ and R⁴ are independently selected from the group consisting of H,optionally substituted C₁₋₁₈ alkyl, optionally substituted C₁₋₁₈ alkoxy,C₁₋₁₈ haloalkyl (e.g., C₁₋₁₈ polyfluoroalkyl), C₂₋₁₈ alkenyl, C₂₋₁₈alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted 5- to10-membered heteroaryl, optionally substituted 5- to 10-memberedheterocyclyl, cyano, halo, nitro, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷,C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂, C(O)NR⁷OR⁸, C(O)C(O)OR⁷ andP(O)(OR⁷)_(2;) and

each R⁷, R⁸, and R⁹ is independently selected from the group consistingof H, optionally substituted C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl,optionally substituted C₆₋₁₀ aryl, optionally substituted 5- to10-membered heteroaryl, and optionally substituted 5- to 10-memberedheterocyclyl.

In some embodiments, R³ and R⁴ is are independently selected from thegroup consisting of H, C₁₋₆ alkoxy, and C₁₋₆ alkyl. In some embodiments,R⁴ is optionally substituted C₁₋₆ alkoxy (e.g., the carbene precursor isa diazoester). For example, R⁴ can be optionally substituted methoxy(e.g., benzyloxy), optionally substituted ethoxy, optionally substitutedn-propoxy, optionally substituted isopropoxy, optionally substitutedn-butoxy, optionally substituted isobutoxy, or optionally substitutedt-butoxy. In some embodiments, R⁴ is C₁₋₆ alkoxy and R³ is H or C₁₋₆alkyl.

In some embodiments, R⁴ is optionally substituted C₁₋₆ alkyl (e.g., thecarbene precursor is a diazoketone). R⁴ can be, for example, methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl,n-pentyl, isopentyl, n-hexyl, or branched hexyl. In some embodiments, R⁴is C₁₋₆ alkoxy and R³ is H or C₁₋₆ alkyl. In some embodiments, R³ is Hand R⁴ is C₁₋₆ alkoxy in carbene precursors according to Formula II. Insome embodiments, the cyclopropene product is a compound according toFormula III as set forth above. In some embodiments, the cyclopropene isselected from:

Cyclopropene products prepared according to the methods described hereincan themselves be used as intermediate compounds for further elaborationvia one more organic transformations. For example, the methods providedherein can further include polymerizing the cyclopropene product. Insome embodiments, the present invention provides a method forring-opening metathesis polymerization (ROMP), as shown in Equation 3.See also, Elling, et al. J. Am. Chem. Soc. , 2015, 137, 9922-9926.

In Equation 3, R¹, R², R³, and R⁴ are independently selected from thegroup consisting of H, optionally substituted C₁₋₁₈ alkyl, C₁₋₁₈haloalkyl (e.g., C₁₋₁₈ polyfluoroalkyl), C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl,optionally substituted C₆₋₁₀ aryl, optionally substituted 5- to10-membered heteroaryl, optionally substituted 5- to 10-memberedheterocyclyl, cyano, halo, nitro, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷,C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂, C(O)NR⁷OR⁸, C(O)C(O)OR⁷ andP(O)(OR⁷)₂.

In some embodiments, the method further comprises converting thecyclopropene product to a cyclopropane. For example, multi-substitutedcyclopropanes can be prepared as shown in Equation 4:

wherein R¹, R², R³, R⁴, and R⁵ are independently selected from the groupconsisting of H, optionally substituted C₁₋₁₈ alkyl, C₁₋₁₈ haloalkyl(e.g., C₁₋₁₈ polyfluoroalkyl), C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, optionallysubstituted C₆₋₁₀ aryl, optionally substituted 5- to 10-memberedheteroaryl, optionally substituted 5- to 10-membered heterocyclyl,cyano, halo, nitro, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷, C(O)SR⁷,C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂, C(O)NR⁷OR⁸, C(O)C(O)OR⁷ and P(O)(OR⁷)₂.

In some embodiments, the method further comprises contacting thecyclopropene product with a diene or a tetrazine under conditionssufficient to form a cycloaddition product. For example, fused ringsystems can be built as shown in Equation 5:

wherein R¹, R², R³, R⁴, and R⁵ are independently selected from the groupconsisting of H, optionally substituted C₁₋₁₈ alkyl, C₁₋₁₈ haloalkyl(e.g., C₁₋₁₈ polyfluoroalkyl), C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, optionallysubstituted C₆₋₁₀ aryl, optionally substituted 5- to 10-memberedheteroaryl, optionally substituted 5- to 10-membered heterocyclyl,cyano, halo, nitro, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷, C(O)SR⁷,C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂, C(O)NR⁷OR⁸, C(O)C(O)OR⁷ and P(O)(OR⁷)₂.

In certain aspects, the present invention provides a method to labelmolecules via inverse Diels Alder cycloadditions, as shown in Equation6. See also, Blackman, et al. J. Am. Chem. Soc., 2008, 130,13518-13519.

In Equation 6, R¹, R², R³, R⁴, and R⁵ are independently selected fromthe group consisting of H, optionally substituted C₁₋₁₈ alkyl, C₁₋₁₈haloalkyl (e.g., C₁₋₁₈ polyfluoroalkyl), C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl,optionally substituted C₆₋₁₀ aryl, optionally substituted 5- to10-membered heteroaryl, optionally substituted 5- to 10-memberedheterocyclyl, cyano, halo, nitro, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷,C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂, C(O)NR⁷OR⁸, C(O)C(O)OR⁷ andP(O)(OR⁷)₂.

C. Bicyclobutane Products

In some embodiments, methods for producing bicyclobutane productscomprise combining an alkyne, a carbene precursor, and a heme protein,homolog thereof, variant thereof, or fragment thereof as describedherein under conditions sufficient to form the bicyclobutane. In someembodiments, the alkyne is a compound according to Formula IV:

wherein

R¹¹ is selected from the group consisting of H, C₁₋₁₈ alkyl, C₁₋₁₈haloalkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, (C₃₋₁₀ cycloalkyl)-L¹¹-, (C₆₋₁₀aryl)-L¹¹-, (5- to 10-membered heteroaryl)-L¹¹-, (5- to 10-memberedheterocyclyl)-L¹¹-, cyano, halo, nitro, N(R¹⁸)₂, B(R¹⁹)₂, Si(R¹⁹)₃,C(O)OR¹⁷, C(O)SR¹⁷, C(O)N(R¹⁷)₂, C(O)R¹⁷, C(O)ON(R¹⁷)₂, C(O)NR¹⁷OR¹⁸,C(O)C(O)OR¹⁷, and P(O)(OR¹⁷)₂;

C₁₋₁₈ alkyl, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, 5- to 10-membered heteroaryl,and 5- to 10-membered heterocyclyl in R¹¹ are optionally andindependently substituted with one or more R^(11a);

each R^(11a) is independently selected from the group consisting ofhalogen, cyano, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ heteroalkyl, C₂₋₆alkynyl, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, and 5- to 10-memberedheterocyclyl;

L¹¹ is selected from the group consisting of a bond and C₁₋₂₀ alkylene;

when L¹¹ is C₂₋₂₀ alkylene, one or more non-adjacent CH₂ groups areoptionally and independently replaced with O, S, or NH;

when L¹¹ is C₃₋₂₀ alkylene, one or more pairs of adjacent CH₂ groups areoptionally and independently replaced with C(O)O or C(O)NH;

R¹² is selected from the group consisting of H, C₁₋₁₈ alkyl, C₁₋₁₈haloalkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, (C₃₋₁₀ cycloalkyl)-L¹²-, (C₆₋₁₀aryl)-L¹²-, (5- to 10-membered heteroaryl) L¹²-, (5- to 10-memberedheterocyclyl)-L¹²-, cyano, halo, nitro, N(R¹⁸)₂, B(R¹⁹)₂, Si(R¹⁹)₃,C(O)OR¹⁷, C(O)SR¹⁷, C(O)N(R¹⁷)₂, C(O)R¹⁷, C(O)ON(R¹⁷)₂, C(O)NR¹⁷OR¹⁸,C(O)C(O)OR¹⁷, and P(O)(OR¹⁷)₂;

C₁₋₁₈ alkyl, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, 5- to 10-membered heteroaryl,and 5- to 10-membered heterocyclyl in R¹² are optionally andindependently substituted with one or more R^(12a);

each R^(12a) is independently selected from the group consisting ofhalogen, cyano, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ heteroalkyl, C₂₋₆alkynyl, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, and 5- to 10-memberedheterocyclyl;

L¹² is selected from the group consisting of a bond and C₁₋₂₀ alkylene;when L¹² is C₂₋₂₀ alkylene, one or more non-adjacent CH₂ groups areoptionally and independently replaced with O, S, or NH;

when L¹² is C₃₋₂₀ alkylene, one or more pairs of adjacent CH₂ groups areoptionally and independently replaced with C(O)O or C(O)NH;

each R¹⁷, R¹⁸, and R¹⁹ is independently selected from the groupconsisting of H, optionally substituted C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl,C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted5- to 10-membered heteroaryl, and optionally substituted 5- to10-membered heterocyclyl.

In some embodiments, and R¹² in alkynes of Formula IV are independentlyselected from H and C₁₋₆ alkyl. R¹¹ and R¹² can be, for example, H,methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, isopentyl, or n-hexyl. In some embodiments, R¹² inalkynes according to Formula IV is H.

In some embodiments, R¹¹ is selected from the group consisting of (C₆₋₁₀aryl)-L¹¹-, (5- to 10-membered heteroaryl)-L¹¹-; C₆₋₁₀ aryl and 5- to10-membered heteroaryl are optionally substituted with one or moreR^(11a); and L¹¹ is a bond.

In some embodiments, R¹¹ is selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkynyl, (C₃₋₁₀ cycloalkyl)-L¹-, (C₆₋₁₀ aryl)-L¹¹-, (5- to10-membered heteroaryl)-L¹¹-, and (5- to 10-membered heterocyclyl)-L¹¹-,each of which is optionally and independently substituted with one ormore R^(11a). In some embodiments, R¹¹ is selected from the groupconsisting of C₁₋₈ alkyl and C₂₋₈ alkynyl, each of which is optionallysubstituted with one or more R^(11a). R^(11a) can be, for example,optionally substituted methyl, optionally substituted ethyl, optionallysubstituted n-propyl, optionally substituted isopropyl, optionallysubstituted n-butyl, optionally substituted isobutyl, optionallysubstituted sec-butyl, optionally substituted tert-butyl, optionallysubstituted n-pentyl, optionally substituted isopentyl, optionallysubstituted n-hexyl, optionally substituted branched hexyl, optionallysubstituted n-heptyl, optionally substituted branched heptyl, optionallysubstituted n-octyl, optionally substituted branched octyl, optionallysubstituted ethynyl, optionally substituted prop-1-yn-1-yl, optionallysubstituted prop-2-yn-1-yl, optionally substituted but-1-yn-1-yl,optionally substituted but-2-yn-1-yl, optionally substitutedbut-3-yn-1-yl, optionally substituted pent-1-yn-1-yl, optionallysubstituted pent-2-yn-1-yl, optionally substituted p ent-3-yn-1-yl,optionally substituted pent-4-yn-1-yl, optionally substitutedhex-1-yn-1-yl, optionally substituted hex-2-yn-1-yl, optionallysubstituted hex-3-yn-1-yl, optionally substituted hex-4-yn-1-yl,optionally substituted hex-5-yn-1-yl, optionally substitutedhept-1-yn-1-yl, optionally substituted hept-2-yn-1-yl, optionallysubstituted hept-3-yn-1-yl, optionally substituted hept-4-yn-1-yl,optionally substituted hept-5-yn-1-yl, optionally substitutedhept-6-yn-1-yl, optionally substituted oct-1-yn-1-yl, optionallysubstituted oct-2-yn-1-yl, optionally substituted oct-3-yn-1-yl,optionally substituted oct-4-yn-1-yl, optionally substitutedoct-5-yn-1-yl, optionally substituted oct-6-yn-1-yl, or optionallysubstituted oct-7-yn-1-yl. Alkynes according to Formula IV havingalkenyl R¹¹ groups, cycloalkyl R¹¹ groups, and the like can also beemployed.

In some embodiments, R¹¹ in alkynes according to Formula IV is selectedfrom the group consisting of C₁₋₆ alkyl and C₂₋₆ alkynyl, each of whichis optionally substituted with one or more R^(11a). In some suchembodiments, R^(11a) is C₁₋₆ alkyl and R^(11a) is selected from halogen(e.g., Cl or Br), —OH, C₁₋₆ alkoxy (e.g., methoxy, ethoxy, n-propoxy, orisopropoxy), and C₁₋₆ heteroalkyl (e.g., methoxymethyl or ethoxymethyl).

In some embodiments, R¹¹ is selected from (C₃₋₁₀ cycloalkyl)-L¹¹-,(C₆₋₁₀ aryl)-L¹¹-, (5- to 10-membered heteroaryl)-L¹¹-, (5- to10-membered heterocyclyl)-L¹¹-, wherein cycloalkyl, aryl, heteroaryl,and heterocyclyl are optionally substituted with one or more R^(11a). Insome embodiments, L¹¹ is a covalent bond. For example, the grouping(C₃₋₁₀ cycloalkyl)-L¹¹- can be optionally substituted cyclopropyl,optionally substituted cyclobutyl, optionally substituted cyclopentyl,optionally substituted cyclohexyl, or the like. In some embodiments, L¹¹is C₁₋₂₀ alkylene (e.g., C₁₋₁₀ alkylene or C₁₋₆ alkylene). For example,the grouping (C₃₋₁₀ cycloalkyl)-L¹¹- can be cyclohexylmethyl,(2-cyclohexyl)eth-1-yl, (3-cyclohexyl)prop-1-yl, or the like. Thegrouping (C₆₋₁₀ aryl)-L¹¹- can be, for example, phenethyl. Cycloalkylgroups, aryl groups, and heterocyclyl groups can be further substitutedwith R^(11a) groups as described above. In some embodiments, R¹¹ iscyclopropyl, cyclobutyl, cyclohexyl, 1,3-dioxolan-2-yl, ortetrahydropyran-1-yl; and R^(11a) is —OH, C₁₋₆ alkoxy (e.g., methoxy,ethoxy, n-propoxy, or isopropoxy), or C₁₋₆ heteroalkyl (e.g.,methoxymethyl or ethoxymethyl).

In some embodiments, L¹¹ is C₁₋₂₀ alkylene where one or morenon-adjacent CH₂ groups are optionally and independently replaced withO, S, or NH. For example, the L¹¹ moiety may have the formula—(CH₂)_(w)(L^(11a) )_(x)(CH₂)_(y)(L^(11a))_(x)(CH₂)_(z)—, where eachL^(11a) is independently O, S, or NH; each subscript x is independently0 or 1; and each of subscripts w, y, and z is a non-zero integer suchthat the sum of subscript w, subscript x, subscript y, and subscript zis less than or equal to 20. In some such embodiments, each L^(11a) is0. In some embodiments, the L¹¹ moiety is —CH₂O(CH₂)₆—.

In some embodiments, L¹¹ is C₃₋₂₀ alkylene wherein one or more pairs ofadjacent CH₂ groups are optionally and independently replaced with C(O)Oor C(O)NH. For example, the L¹¹ moiety may have the formula—(CH₂)_(q)(L^(11b))_(r)(CH₂)_(s)(L^(11b))_(t)(CH₂)_(u)—, where eachL^(11b) is C(O)O or C(O)NH; subscript r and subscript t areindependently 0 or 1; and each of subscripts q, s, and u is a non-zerointeger such that: 1) the sum of subscript q, subscript s, and subscriptu is less than or equal to 20 when subscript r and subscript t are 0; 2)the sum of subscript q, subscript s, and subscript u is less than orequal to 18 when the sum of subscript r and subscript t is 1; or 3) thesum of subscript q, subscript s, and subscript u is less than or equalto 16 when the sum of subscript r and subscript t is 2. In someembodiments, the L¹¹ moiety is C(O)O(CH₂)₆—. L¹¹ groups having both oneor more L^(11a) moieties (i.e., O, S, or NH) and one or more L^(11b)moieties (i.e., C(O)O or C(O)NH) can also be employed.

In some embodiments, each R^(11a) is independently selected from thegroup consisting of halogen, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆heteroalkyl (e.g., hydroxymethyl), and C₂₋₆ alkynyl. In someembodiments, the alkyne for forming bicyclobutane products is selectedfrom:

In some embodiments, the alkyne for forming bicyclobutane products isselected from:

In some embodiments, the alkyne for forming bicyclobutane products isselected from:

Any suitable carbene precursor (e.g., an amine, an azide, a hydrazine, ahydrazone, an epoxide, a diazirene, or a diazo compound) can be used inthe methods. In some embodiments, the carbene precursor is a diazocompound such as a compound according to Formula V:

wherein

R¹³ and R¹⁴ are independently selected from the group consisting of H,optionally substituted C₁₋₁₈ alkyl, optionally substituted C₁₋₁₈ alkoxy,C₁₋₁₈ haloalkyl (e.g., C₁₋₁₈ polyfluoroalkyl), C₂₋₁₈ alkenyl, C₂₋₁₈alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted 5- to10-membered heteroaryl, optionally substituted 5- to 10-memberedheterocyclyl, cyano, halo, nitro, N(R¹⁸)₂, B(R¹⁹)₂, Si(R¹⁹)₃, C(O)OR¹⁷,C(O)SR¹⁷, C(O)N(R¹⁷)₂, C(O)R¹⁷, C(O)ON(R¹⁷)₂, C(O)NR¹⁷OR¹⁸, C(O)C(O)OR¹⁷and P(O)(OR¹⁷)₂; and each R¹⁷, R¹⁸, and R¹⁹ is independently selectedfrom the group consisting of H, optionally substituted C₁₋₁₈ alkyl,C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl,optionally substituted 5- to 10-membered heteroaryl, and optionallysubstituted 5- to 10-membered heterocyclyl.

In some embodiments, R¹³ and R¹⁴ are independently selected from thegroup consisting of H, C₁₋₆ alkoxy, and C₁₋₆ alkyl. In some embodiments,R¹⁴ is optionally substituted C₁₋₆ alkoxy (e.g., the carbene precursoris a diazoester). For example, R¹⁴ can be optionally substituted methoxy(e.g., benzyloxy), optionally substituted ethoxy, optionally substitutedn-propoxy, optionally substituted isopropoxy, optionally substitutedn-butoxy, optionally substituted isobutoxy, or optionally substitutedt-butoxy. In some embodiments, R¹⁴ is C₁₋₆ alkoxy and R¹³ is H or C₁₋₆alkyl.

In some embodiments, R¹⁴ is optionally substituted C₁₋₆ alkyl (e.g., thecarbene precursor is a diazoketone). R¹⁴ can be, for example, methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl,n-pentyl, isopentyl, n-hexyl, or branched hexyl. In some embodiments,R¹⁴ is C₁₋₆ alkoxy and R¹³ is H or C₁₋₆ alkyl.

In some embodiments, R¹³ is H and R¹⁴ is C₁₋₆ alkoxy in carbeneprecursors according to Formula V. In some embodiments, the product is abicyclobutane according to Formula VI as set forth above. In someembodiments, the bicyclobutane is selected from:

Bicyclobutane products prepared according to the methods describedherein can themselves be used as intermediate compounds for furtherelaboration via one more organic transformations, as described abovewith respect to cyclopropenes. In certain aspects, the present inventionprovides a method for producing polyester by using the bicyclobutaneproduct according to Equation 7:

wherein R¹, R², R³, R⁴, R⁵⁰, R⁶⁰, R⁷⁰, and R⁸⁰ are independentlyselected from the group consisting of H, optionally substituted C₁₋₁₈alkyl, C₁₋₁₈ haloalkyl (e.g., C₁₋₁₈ polyfluoroalkyl), C₂₋₁₈ alkenyl,C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted5- to 10-membered heteroaryl, optionally substituted 5- to 10-memberedheterocyclyl, cyano, halo, nitro, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷,C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂, C(O)NR⁷OR⁸, C(O)C(O)OR⁷ andP(O)(OR⁷)₂.

In certain aspects, the present invention provides a method forsynthesizing highly functionalized chiral cyclobutane-containingmolecules via strain-release amination, as shown in Equation 8. Seealso, Gianatassio, et al. Science 2016, 351, 241-246.

In Equation 8, R¹, R², R³, R⁴, R⁵, and R⁶ are independently selectedfrom the group consisting of H, optionally substituted C₁₋₁₈ alkyl,C₁₋₁₈ haloalkyl (e.g., C₁₋₁₈ polyfluoroalkyl), C₂₋₁₈ alkenyl, C₂₋₁₈alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted 5- to10-membered heteroaryl, optionally substituted 5- to 10-memberedheterocyclyl, cyano, halo, nitro, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷,C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂, C(O)NR⁷01e, C(O)C(O)OR⁷ andP(O)(OR⁷)₂.

Accordingly, some of the embodiments disclosed herein provide methodswhich further include hydrolyzing the bicyclobutane to form adicarboxylate-substituted bicyclobutane. In some embodiments, the methodfurther comprises contacting the dicarboxylate-substituted bicyclobutanewith an amine under conditions sufficient to form a diamido-substitutedbicyclobutane. In some embodiments, the method further comprisescontacting the dicarboxylate-substituted bicyclobutane with a diol underconditions sufficient to form a polyester.

In certain aspects, the present invention provides a method forproducing functionalized polybenzvalenes from bicyclobutane productsaccording to Equation 9. Functionalized polybenzvalenes contains highstrain-energies and have potential applications in propulsiontechnologies. (See, e.g., Agrawal, (2010). High energy materials:propellants, explosives and pyrotechnics. John Wiley & Sons.)Non-conjugated polybenzvalene polymers could also isomerize toconjugated conductive polyacetylenes without extrusion of molecularfragments as shown in Equation 10. (See, Swager, et al. J. Am. Chem.Soc., 1988, 110(9), 2973-2974.) This feature makes polybenzvaleneshighly desirable for producing conductive polymers with highly orderedmorphologies. Furthermore, as this isomerization process can be promotedchemically, photochemically, mechanically, thermally, and by transitionmetals, polybenzvalene polymers have huge potentials asstimuli-responsive materials. (See, Chen, et al. Science, 2017, 357,475-479.)

In Equation 9 and Equation 10, R¹, R², R³, and R⁴ are independentlyselected from the group consisting of H, optionally substituted C₁₋₁₈alkyl, C₁₋₁₈ haloalkyl (e.g., C₁₋₁₈ polyfluoroalkyl), C₂₋₁₈ alkenyl,C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted5- to 10-membered heteroaryl, optionally substituted 5- to 10-memberedheterocyclyl, cyano, halo, nitro, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷,C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂, C(O)NR⁷OR⁸, C(O)C(O)OR⁷ andP(O)(OR⁷)₂.

Accordingly, some of the embodiments disclosed herein provide methodswhich further include reducing the bicyclobutane to form adihydroxy-substituted bicyclobutane. In some embodiments, the methodsfurther include converting the dihydroxy-substituted bicyclobutane to apolybenzvalene or a polyacetylene.

In certain aspects, the present invention provides a method forincorporating bicyclobutane groups into molecules via click chemistry,as shown in Equation 11.

In Equation 11, R¹, R², R³, R⁴, R⁵, and R⁶ are independently selectedfrom the group consisting of H, optionally substituted C₁₋₁₈ alkyl,C₁₋₁₈ haloalkyl (e.g., C₁₋₁₈ polyfluoroalkyl), C₂₋₁₈ alkenyl, C₂₋₁₈alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted 5- to10-membered heteroaryl, optionally substituted 5- to 10-memberedheterocyclyl, cyano, halo, nitro, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷,C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂, C(O)NR⁷OR⁸, C(O)C(O)OR⁷ andP(O)(OR⁷)₂, and L is a linking moiety (e.g., a linking moiety L¹ or L¹¹as described above).

Accordingly, some of the embodiments disclosed herein provide methodswherein the bicyclobutane comprises an alkyne moiety and the methodfurther comprises contacting the alkyne moiety with an azide underconditions sufficient to form a triazole moiety.

The alkyne reagents, carbene precursor reagents, and strained carbocycleproducts described herein can be further substituted. A compoundaccording to Formula I, Formula Ia, or Formula III may contain, forexample, an optionally substituted R¹ group or an optionally substitutedR² group, while a compound according to Formula II or Formula III maycontain an optionally substituted R³ group, one or more optionallysubstituted R⁴ groups. A compound according to Formula IV or Formula VImay contain, for example, an optionally substituted R¹¹ group or anoptionally substituted R¹² group, while a compound according to FormulaV or Formula VI may contain an optionally substituted R¹³ group, one ormore optionally substituted R¹⁴ groups. In general, the term“substituted,” whether preceded by the term “optionally” or not, meansthat one or more hydrogens of the designated moiety are replaced with asuitable substituent. Unless otherwise indicated, an “optionallysubstituted” group may have a suitable substituent at each substitutableposition of the group, and when more than one position in any givenstructure is substituted with more than one substituent selected from aspecified group, the substituent may be either the same or different atevery position. Combinations of substituents are generally those thatresult in the formation of stable or chemically feasible compounds. Theterm “stable,” as used herein, refers to compounds that are notsubstantially altered when subjected to conditions to allow for theirproduction, detection, and, in certain embodiments, their recovery,purification, and use for one or more of the purposes disclosed herein.In general, “substituted,” as used herein, does not encompassreplacement and/or alteration of a key functional group by which amolecule is identified, e.g., such that the “substituted” functionalgroup becomes, through substitution, a different functional group. Forexample, a “substituted phenyl” group must still comprise the phenylmoiety and cannot be modified by substitution, in this definition, tobecome, e.g., a cyclohexyl group.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(α); —(CH₂)₀₋₄OR^(α); —O(CH₂)₀₋₄R^(α); —O—(CH₂)₀₋₄C(O)OR^(α);—(CH₂)₀₋₄CH(OR^(α))₂; —(CH₂)₀₋₄SR^(α); —(CH₂)₀₋₄Ph, wherein Ph is phenylwhich may be substituted with R^(α); —(CH₂)₀₋₄O(CH₂)₀₋₁phenyl, whichphenyl may be substituted with R^(α); —CH═CHPh, wherein Ph is phenylwhich may be substituted with R^(α); —(CH₂)₀₋₄O(CH₂)₀₋₁-Py, wherein Pyis pyridyl which may be substituted with R^(α); —NO₂; —CN; —N₃;—(CH₂)₀₋₄N(R^(α))₂; —(CH₂)₀₋₄N(H^(α))C(O)R^(α); —N(R^(α))C(S)R^(α);—(CH₂)₀₋₄N(R^(α))C(O)NR^(α) ₂; —N(R^(α))C(S)NR^(α) ₂;—(CH₂)₀₋₄N(H^(α))C(O)OR^(α); —N(R^(α))N(R^(α))C(O)R^(α);—N(R^(α))N(R^(α))C(O)NR^(α) ₂; —N(R^(α))N(R^(α))C(O)OR^(α);—(CH₂)₀₋₄C(O)R^(α); —C(S)R^(α); —(CH₂)₀₋₄C(O)OR^(α);—(CH₂)₀₋₄C(O)SR^(α); —(CH₂)₀₋₄C(O)OSiR^(α) ₃; —(CH₂)₀₋₄OC(O)R^(α);—OC(O)(CH₂)₀₋₄SR—SC(S)SR^(α); —(CH₂)₀₋₄SC(O)R^(α); —(CH₂)₀₋₄C(O)NR^(α)₂; —C(S)NR^(α) ₂, —C(S)SR^(α); —SC(S)SR^(α), —(CH₂)₀₋₄OC(O)NR^(α) ₂;—C(O)N(OR^(α))R^(α); —C(O)C(O)R^(α); —C(O)CH₂C(O)R^(α);—C(NOR^(α))R^(α); —(CH₂)₀₋₄SSR^(α); —(CH₂)₀₋₄S(O)₂R^(α);—(CH₂)₀₋₄S(O)₂OR^(α); —(CH₂)₀₋₄OS(O)₂R^(α); —S(O)₂NR^(α) ₂;—(CH₂)₀₋₄S(O)R^(α); —N(R^(α))S(O)₂NR^(α) ₂; —N(R^(α))S(O)₂R^(α);—N(OR^(α))R^(α); —C(NH)NR^(αhd 2); —P(O)₂R^(α); —P(O)R^(α) ₂;—OP(O)R^(α) ₂; —OP(O)(OR^(α))₂; SiR^(α) ₃; —(C₁₋₄ straight orbranched)alkylene)-O—N(R^(α))₂; or —(C₁₋₄ straight orbranched)alkylene)C(O)O—N(R^(α))₂. Each R^(α)is independently hydrogen;C₁₋₆ alkyl; —CH₂Ph, —O(CH₂)₀₋₁Ph; —CH₂-(5- to 6-membered heteroaryl);C₃₋₈ cycloalkyl; C₆₋₁₀ aryl; 4- to 10-membered heterocyclyl; or 6- to10-membered heteroaryl; and each R^(α)may be further substituted asdescribed below.

Suitable monovalent substituents on IV are independently halogen,—(CH₂)₀₋₂R^(β); —(CH₂)₀₋₂OH; —(CH₂)₀₋₂OR^(β); —(CH₂)₀₋₂CH(OR^(β))₂; —CN;—N₃; —(CH₂)₀₋₂C(O)R^(β); —(CH₂)₀₋₂C(O)OH; —(CH₂)₀₋₂C(O)OR^(β);—(CH₂)₀₋₂SR^(β); —(CH₂)₀₋₂SH; —(CH₂)₀₋₂NH₂; —(CH₂)₀₋₂NHR^(β);—(CH₂)₀₋₂NR^(β) ₂; —NO₂; SiR^(β) ₃; —OSiR^(β) ₃; —C(O)SR^(β); —(C₁₋₄straight or branched alkylene)-C(O)OR^(β); or —SSR^(β) ₂; wherein eachR^(β) is independently selected from C₁₋₄ alkyl; —CH₂Ph; —O(CH₂)₀₋₁Ph;C₃₋₈ cycloalkyl; C₆₋₁₀ aryl; 4- to 10-membered heterocyclyl; or 6- to10-membered heteroaryl. Suitable divalent substituents on a saturatedcarbon atom of R^(α) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O; ═S; ═NNR^(γ)₂; ═NNHC(O)R^(γ); ═NNHC(O)OR^(γ); ═NNHS(O)₂R^(γ); ═NR^(γ); ═NOR^(γ);—O(C(R^(γ) ₂))₂₋₃O—; or —S(C(R^(γ) ₂))₂₋₃S—; wherein each independentoccurrence of R^(γ) is selected from hydrogen; C₁₋₆ alkyl, which may besubstituted as defined below; C₃₋₈ cycloalkyl; C₆₋₁₀ aryl; 4- to10-membered heterocyclyl; or 6- to 10-membered heteroaryl. Suitabledivalent substituents that are bound to vicinal substitutable carbons ofan “optionally substituted” group include: —O(CR^(β) ₂)₂₋₃O—; whereineach independent occurrence of R¹ is selected from hydrogen; C₁₋₆ alkylwhich may be substituted as defined below; C₃₋₈ cycloalkyl; C₆₋₁₀ aryl;4- to 10-membered heterocyclyl; or 6- to 10-membered heteroaryl.

Suitable substituents on the alkyl group of R^(γ)include halogen;—R^(δ); —OH; —OR^(δ); —CN; —C(O)OH; —C(O)OR^(δ); —NH₂; —NHR^(δ); —NR^(δ)₂; or —NO₂; wherein each R^(d) is independently C₁₋₄ alkyl; —CH₂Ph;—O(CH₂)₀₋₁Ph; 4- to 10-membered heterocyclyl; or 6- to 10-memberedheteroaryl.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include R^(ε); —NR^(ε) ₂; —C(O)R^(ε); —C(O)OR^(ε);—C(O)C(O)R^(ε); —C(O)CH₂C(O)R^(ε); —S(O)₂R^(ε); —S(O)₂NR^(ε) ₂;—C(S)NR^(ε) ₂; —C(NH)NR^(ε) ₂; or —N(R^(ε))S(O)₂R^(ε); wherein eachR^(ε) is independently hydrogen; C₁₋₆ alkyl which may be substituted asdefined below; C₃₋₈ cycloalkyl; C₆₋₁₀ aryl; 4- to 10-memberedheterocyclyl; or 6- to 10-membered heteroaryl.

Suitable substituents on the alkyl group of R^(ε) are independentlyhalogen; —R^(δ); —OH; δOR^(δ); —CN; —C(O)OH; —C(O)OR^(δ); —NH₂;—NHR^(δ); —NR^(δ) ₂; or —NO₂; wherein each R^(δ) is independently C₁₋₄alkyl; —CH₂Ph; —O(CH₂)₀₋₁Ph; C₆₋₁₀ aryl; 4- to 10-membered heterocyclyl;or 6- to 10-membered heteroaryl.

D. Reaction Conditions for Enzyme-Catalyzed Carbocycle Formation

The enzymes of the present invention can be used in purified form,partially purified form, or as whole-cell (e.g., bacterial) catalysts,without purification. Many alkyne substrates and carbene precursors canenter E. coli cells and interact with the enzymes inside the cells,where the reaction takes place. Thus strained rings can be made in aprocess wherein intact or partially permeabilized cells expressing theenzyme catalyst are suspended in buffer and combined with alkyne andcarbene precursor (dissolved in appropriate solvent or in a form ofsuspension) and allowed to react. The process can also use purified orpartially purified protein in place of whole bacterial cells. Otherprocesses can involve changing contacting conditions (e.g., maintainingthe catalyst in a compartment such as behind a filter membrane or bagthrough which reactants and products can pass or immobilizing thecatalyst in some other way). One skilled in the art will be able toidentify appropriate processing conditions for a given set of substratesand catalysts.

The methods of the invention include forming reaction mixtures thatcomprise an alkyne, a carbene precursor, and a heme protein, fragmentthereof, homolog thereof, or variant thereof as described above. In someembodiments, the method is carried out in vitro. In other embodiments,the heme protein is localized within a whole cell and the method iscarried out in vivo. In some embodiments, the heme protein is expressedin a bacterial, archaeal, yeast or fungal host organism. In someembodiments, the method is carried out under anaerobic conditions. Inother embodiments, the process is carried out under aerobic conditions.

The heme proteins, fragments thereof, homologs thereof, or variantsthereof can be, for example, purified prior to addition to a reactionmixture or secreted by a cell present in the reaction mixture. Thereaction mixture can contain a cell lysate including the heme protein,fragment thereof, homolog thereof, or variant thereof, as well as otherproteins and other cellular materials. Alternatively, a heme protein,fragment thereof, homolog thereof, or variant thereof can catalyze thereaction within a cell expressing the heme protein, fragment thereof,homolog thereof, or variant thereof. Any suitable amount of hemeprotein, fragment thereof, homolog thereof, or variant thereof can beused in the methods of the invention. In general, the reaction mixtureswill contain from about 0.01 mol % to about 10 mol % heme protein withrespect to the carbene precursor (e.g., diazo reagent) and/or alkyne.The reaction mixtures can contain, for example, from about 0.01 mol % toabout 0.1 mol % heme protein, or from about 0.1 mol % to about 1 mol %heme protein, or from about 1 mol % to about 10 mol % heme protein. Thereaction mixtures can contain from about 0.05 mol % to about 5 mol %heme protein, or from about 0.05 mol % to about 0.5 mol % heme protein.The reaction mixtures can contain about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, or about 1 mol % heme protein.

The concentration of the alkyne and the carbene precursor (e.g., a diazoreagent) are typically in the range of from about 100 μM to about 1 M.The concentration can be, for example, from about 100 μM to about 1 mM,or about from 1 mM to about 100 mM, or from about 100 mM to about 500mM, or from about 500 mM to 1 M. The concentration can be from about 500μM to about 500 mM, 500 μM to about 50 mM, or from about 1 mM to about50 mM, or from about 15 mM to about 45 mM, or from about 15 mM to about30 mM. The concentration of alkyne or carbene precursor can be, forexample, about 100, 200, 300, 400, 500, 600, 700, 800, or 900 μM. Theconcentration of alkyne or carbene precursor can be about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mM.

Reaction mixtures can contain additional reagents. As non-limitingexamples, the reaction mixtures can contain buffers (e.g., M9-N buffer,2-(N-morpholino)ethanesulfonic acid (MES),2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES),3-morpholinopropane-1-sulfonic acid (MOPS),2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), potassium phosphate,sodium phosphate, phosphate-buffered saline, sodium citrate, sodiumacetate, and sodium borate), cosolvents (e.g., dimethylsulfoxide,dimethylformamide, ethanol, methanol, isopropanol, glycerol,tetrahydrofuran, acetone, acetonitrile, and acetic acid), salts (e.g.,NaCl, KCl, CaCl₂, and salts of Mn²⁺ and Mg²⁺), denaturants (e.g., ureaand guanadinium hydrochloride), detergents (e.g., sodium dodecylsulfateand Triton-X 100), chelators (e.g., ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA),2-({2-[Bis(carboxymethyl)amino]ethyl} (carboxymethyl)amino)acetic acid(EDTA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid(BAPTA)), sugars (e.g., glucose, sucrose, and the like), and reducingagents (e.g., sodium dithionite, NADPH, dithiothreitol (DTT),β-mercaptoethanol (BME), and tris(2-carboxyethyl)phosphine (TCEP)).Buffers, cosolvents, salts, denaturants, detergents, chelators, sugars,and reducing agents can be used at any suitable concentration, which canbe readily determined by one of skill in the art. In general, buffers,cosolvents, salts, denaturants, detergents, chelators, sugars, andreducing agents, if present, are included in reaction mixtures atconcentrations ranging from about 1μM to about 1 M. For example, abuffer, a cosolvent, a salt, a denaturant, a detergent, a chelator, asugar, or a reducing agent can be included in a reaction mixture at aconcentration of about 1μM, or about 10 μM, or about 100 μM, or about 1mM, or about 10 mM, or about 25 mM, or about 50 mM, or about 100 mM, orabout 250 mM, or about 500 mM, or about 1 M. In some embodiments, areducing agent is used in a sub-stoichiometric amount with respect tothe olefin substrate and the diazo reagent. Cosolvents, in particular,can be included in the reaction mixtures in amounts ranging from about1% v/v to about 75% v/v, or higher. A cosolvent can be included in thereaction mixture, for example, in an amount of about 5, 10, 20, 30, 40,or 50% (v/v).

Reactions are conducted under conditions sufficient to catalyze theformation of a cyclopropene product or a bicyclobutane product. Thereactions can be conducted at any suitable temperature. In general, thereactions are conducted at a temperature of from about 4° C. to about40° C. The reactions can be conducted, for example, at about 25° C. orabout 37° C. The heme proteins or cells expressing or containing theheme proteins can be heat treated. In some embodiments, heat treatmentoccurs at a temperature of about 75° C. The reactions can be conductedat any suitable pH. In general, the reactions are conducted at a pH offrom about 6 to about 10. The reactions can be conducted, for example,at a pH of from about 6.5 to about 9 (e.g., about 6.5, 6.6, 6.7, 6.8,6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2,8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0). The reactions can beconducted for any suitable length of time. In general, the reactionmixtures are incubated under suitable conditions for anywhere betweenabout 1 minute and several hours. The reactions can be conducted, forexample, for about 1 minute, or about 5 minutes, or about 10 minutes, orabout 30 minutes, or about 1 hour, or about 2 hours, or about 4 hours,or about 8 hours, or about 12 hours, or about 24 hours, or about 48hours, or about 72 hours. The reactions can be conducted for about 1 to4 hours (e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, or 4 hours). Reactions can be conducted under aerobicconditions or anaerobic conditions. Reactions can be conducted under aninert atmosphere, such as a nitrogen atmosphere or argon atmosphere. Insome embodiments, a solvent is added to the reaction mixture. In someembodiments, the solvent forms a second phase, and the carbene insertioninto carbon-carbon bonds occurs in the aqueous phase. In someembodiments, the heme protein, fragment thereof, variant thereof, orhomolog thereof, is located in the aqueous layer whereas the substratesand/or products occur in an organic layer. Other reaction conditions maybe employed in the methods of the invention, depending on the identityof a particular heme protein, alkyne, or carbene precursor (e.g., diazoreagent).

Reactions can be conducted in vivo with intact cells expressing a hemeenzyme of the invention. The in vivo reactions can be conducted with anyof the host cells used for expression of the heme enzymes, as describedherein. A suspension of cells can be formed in a suitable mediumsupplemented with nutrients (such as mineral micronutrients, glucose andother fuel sources, and the like). Product yields from reactions in vivocan be controlled, in part, by controlling the cell density in thereaction mixtures. Cellular suspensions exhibiting optical densitiesranging from about 0.1 to about 50 at 600 nm can be used for thecyclopropene- and bicyclobutane-forming reactions. Other densities canbe useful, depending on the cell type, specific heme proteins, or otherfactors.

The methods of the invention can be assessed in terms of thediastereoselectivity and/or enantioselectivity of carbene insertion intoalkyne carbon-carbon bonds—that is, the extent to which the reactionproduces a particular isomer, whether a diastereomer or enantiomer. Aperfectly selective reaction produces a single isomer, such that theisomer constitutes 100% of the product. As another non-limiting example,a reaction producing a particular enantiomer constituting 90% of thetotal product can be said to be 90% enantioselective. A reactionproducing a particular diastereomer constituting 30% of the totalproduct, meanwhile, can be said to be 30% diastereoselective.

In general, the methods of the invention include reactions that are fromabout 1% to about 99% diastereoselective. The reactions are from about1% to about 99% enantioselective. The reaction can be, for example, fromabout 10% to about 90% diastereoselective, or from about 20% to about80% diastereoselective, or from about 40% to about 60%diastereoselective, or from about 1% to about 25% diastereoselective, orfrom about 25% to about 50% diastereoselective, or from about 50% toabout 75% diastereoselective. The reaction can be about 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, orabout 95% diastereoselective. The reaction can be from about 10% toabout 90% enantioselective, from about 20% A to about 80%enantioselective, or from about 40% to about 60% enantioselective, orfrom about 1% to about 25% enantioselective, or from about 25% to about50% enantioselective, or from about 50% to about 75% enantioselective.The reaction can be about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or about 95% enantioselective.Accordingly some embodiments of the invention provide methods whereinthe reaction is at least 30% to at least 90% diastereoselective. In someembodiments, the reaction is at least 30% to at least 90%enantioselective. Preferably, the reaction is at least 80% (e.g., atleast about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) enantioselective. Morepreferably, the reaction is at least 90% (e.g., at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) enantioselective.

IV. EXAMPLES

The present invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes, and are not intended to limit the invention in any manner.Those of skill in the art will readily recognize a variety ofnoncritical parameters which can be changed or modified to yieldessentially the same results.

Example 1 Enzymatic Cyclopropenation to Yield Compounds of Formula IIIUsing P450_(BM3) Variants In Vivo

P450_(BM3) expression. 45 ml Hyperbroth (100 μg/ml ampicillin) wasinoculated with an overnight culture of 5 ml LB (100 μg/ml ampicillin).The overnight culture contained recombinant E. coli BL21-DE3 cellsharboring a pET22 plasmid, encoding the P450_(BM3) variant under thecontrol of the T7 promoter, and the P450_(BM3) maturation (ccm) operonunder the control of a tet promoter, respectively. The cultures wereshaken at 220 rpm at 37° C. for approximately 2.5 h. The flaskcontaining the cells was placed on ice for 20 min. The incubatortemperature was reduced to 20° C., maintaining the 130 rpm shake rate.Cultures were induced by adding IPTG and aminolevulinic acid to a finalconcentration of 0.5 mM and 0.5 mM respectively. The cultures wereallowed to continue for another 18-22 hours at this temperature andshake rate. Cells were harvested by centrifugation (4° C., 6 min,4,500×g) to produce a cell pellet.

Preparation of whole cell catalysts. To prepare whole cells forcatalysis, the cell pellet prepared in the previous paragraph wasresuspended in M9-N minimal media (M9 media without ammonium chloride)to an optical density (OD₆₀₀) of 60. The cell suspension was used as thecatalyst.

Purification of P450_(BM3). To prepare purified proteins, the cellpellet prepared as described above was stored at −20° C. or belowovernight. Frozen cells were resuspended in buffer A (25 mM tris, 20 mMimidazole, 200 mM NaCl, pH 7.5, 4 ml/g of cell wet weight), loaded with300 μg/ml hemin, and lysed by sonication. To pellet insoluble material,lysates were centrifuged (20,000×g, 15 min, 4° C.). Proteins wereexpressed in a construct containing a 6×-His tag and purified using anickel NTA column (1 ml HisTrap HP, GE Healthcare, Piscataway, N.J.)using an AKTAxpress purifier FPLC system (GE Healthcare). P411 enzymeswere eluted on a linear gradient from 100% buffer A/0% buffer B (25 mMtris, 300 mM imidazole, 200 mM NaCl, pH 7.5) to 100% buffer B over 10column volumes. Fractions containing P411 enzymes were pooled,concentrated, and subjected to three exchanges of phosphate buffer (0.1M KPi, pH 8.0) to remove excess salt and imidazole. Concentratedproteins were aliquotted, flash-frozen on powdered dry ice, and storedat −20° C.

Small-scale cyclopropenation reactions in whole-cell suspension underanaerobic conditions. Small-scale (400 μL) reactions were carried out in2 ml glass crimp vials (Agilent Technologies, San Diego, Calif.). Cellsuspension (OD₆₀₀=60, 340 μL) was added to an unsealed crimp vial beforecrimp sealing with a silicone septum. The headspace of the vial wasflushed with argon for 10 min (no bubbling). A solution of D-glucose (40μL, 250 mM) was added, followed by a solution of alkyne of formula I (10μl, 400 mM in EtOH; for example, 4-phenylbutyne) and a solution of diazoreagent of formula II (10 μl, 400 mM in EtOH; for example, ethyldiazoethanoate or EDA). The reaction vial was left to shake on a plateshaker at 640 rpm for 8 h at room temperature. To quench the reaction,the vial was uncapped and a 1:1 mixture of ethylacetate/cyclohexane (1ml) was added, followed by 1,2,3-trimethoxybenzene (20 μl, 20 mM intoluene) as an internal standard. The mixture was transferred to a 1.5ml Eppendorf tube and vortexed and centrifuged (14000×rcf, 5 min). Theorganic layer was analyzed by gas chromatography (GC), gaschromatography-mass spectrometry (GC-MS), supercritical fluidchromatography (SFC) or normal-phase high performance liquidchromatography (HPLC).

The results of the small scale reactions are shown in FIG. 1 anddemonstrate that P450_(BM3) and variants thereof are capable ofcatalyzing the cyclopropenation to give product of Formula III with highselectivity. Specifically, the best variant P4 found in the initialscreen of P450_(BM3) variants encoded the mutations V87A, A328, A268Gbased on P411_(BM3) “1263F” variant (mutations in P4 relative towild-type P450_(BM3): V78A, F87A, P142S, T1751, A184V, S226R, H236Q,E252G, I263F, T268G, A290V, A328V, L353V, I366V, C400S, T438S, E442K;mutations in P411_(BM3) “1263F” relative to wild-type P450_(BM3): V78A,F87V, P142S, T175I, A184V, S226R, H236Q, E252G, 1263F, T268A, A290V,L353V, I366V, C400S, T438S, E442K), which catalyzed the desired reactionwith 450 TTN and 91% ee. This can be improved by further engineering, ifdesired.

The results of the small-scale reactions are shown in FIG. 2 anddemonstrate that P4 (mutations in P4 relative to wild-type P450_(BM3):V78A, F87A, P142S, T1751, A184V, S226R, H236Q, E252G, I263F, T268G,A290V, A328V, L353V, 1366V, C400S, T438S, E442K) and variants thereofare capable of catalyzing the cyclopropenation to give product ofFormula III with high selectivity. Specifically, the best variant forthe synthesis of one cyclopropene enantiomer found in the initial screenof P4 variants encoded the mutations A87W, V328Y, L437G based on P4variant, which catalyzed the desired reaction with 2990 TTN and 99.6%ee. The best variant for the synthesis of the other cyclopropeneenantiomer found in the initial screen of P4 variants encoded themutations S72W, A87F, F261G, T327P, L437F based on P4 variant, whichcatalyzed the desired reaction with 2140 TTN and −96.2% ee. These twovariants can be improved by further engineering, if desired.

Example 2 Enzymatic Bicyclobutanation to Yield Compounds of Formula VIUsing P450_(BM3) Variants In Vivo

P450_(BM3) expression. The procedures for P450_(BM3) expression wereidentical to those described in Example 1.

Preparation of whole cell catalysts. The procedures for whole cellcatalyst preparation were identical to those described in Example 1,except OD₆₀₀=30.

Small-scale bicyclobutanation reactions in whole-cell suspension underanaerobic conditions. Small-scale (400 μL) reactions were carried out in2 ml glass crimp vials (Agilent Technologies, San Diego, Calif.). Cellsuspension (OD₆₀₀=30, 340 μL) was added to an unsealed crimp vial beforecrimp sealing with a silicone septum. The headspace of the vial wasflushed with argon for 10 min (no bubbling). A solution of D-glucose (40μL, 250 mM) was added, followed by a solution of alkyne of Formula IV(10 μl, 400 mM in EtOH; for example, phenylacetylene) and a solution ofdiazo reagent of Formula V (10 μl, 400 mM in EtOH; for example, ethyldiazoethanoate or EDA). The reaction vial was left to shake on a plateshaker at 640 rpm for 8 h at room temperature. To quench the reaction,the vial was uncapped and a 1:1 mixture of ethylacetate/cyclohexane (1ml) was added, followed by 1,2,3-trimethoxybenzene (20 μl, 20 mM intoluene) as an internal standard. The mixture was transferred to a 1.5ml Eppendorf tube and vortexed and centrifuged (14000× ref, 5 min). Theorganic layer was analyzed by gas chromatography (GC), gaschromatography-mass spectrometry (GC-MS), supercritical fluidchromatography (SFC) or normal-phase high performance liquidchromatography (HPLC).

The results of the small scale reactions are shown in Table 1A anddemonstrate that P450_(BM3) and variants thereof are capable ofcatalyzing the bicyclobutanation to give products of Formula VI withhigh selectivity. Specifically, the best variant E10) found in theinitial screen of P450_(BM3) variants encoded the mutations A78V, F263Lbased on P411_(BM3) “A82L” variant (mutations in E10 relative towild-type P450_(BM3): A82L, F87A, P142S, T175I, A184V, S226R, H236Q,E252G, I263L, T268G, A290V, A328V, L353V, I366V, C400S, T438S, E442K;mutations in P411_(BM3) “A82L” relative to wild-type P450_(BM3): V78A,A82L, F87A, P142S, T1751, A184V, S226R, H236Q, E252G, I263F, T268G,A290V, A328V, L353V, I366V, C400S, T438S, E442K), which catalyzed thedesired reaction with 1380 TTN. This can be improved by furtherengineering, if desired.

TABLE 1A

P411BM3 variant Result “1263F” A328V  3% yield “1263F” A328V V87A  7%yield “1263F” A328V V87A A268G 10% yield “1263F” A328V V87A A268G A82L34% yield (280 TTN) “1263F” A328V V87A A268G A82I  8% yield “A82L” A78V34% yield (360 TTN) “A82L” A78V F263L 43% yield (530 TTN) “A82L” F263YA78L A74G T327I  9% yield

The results of the small scale reactions are shown in FIG. 3 anddemonstrate that E10 (mutations in E10 relative to wild-type P450_(BM3):A82L, F87A, P142S, T175I, A184V, S226R, H236Q, E252G, I263L, T268G,A290V, A328V, L353V, I366V, C400S, T438S, E442K) and variants thereofare capable of catalyzing the bicyclobutanation to give product offormula IV with high selectivity. Specifically, the best variant forbicyclobutanation found in the initial screen of E10 variants encodedthe mutations V78F, S438A based on E10 variant, which catalyzed thedesired reaction with 1880 TTN. This can be improved by furtherengineering, if desired.

Example 3 Preparation of Bicyclobutane-Dicarboxylic Acids and PolyesterSynthesis Using Diacids and Diols

Aliphatic polyesters with enhanced hydrophilicity and less toxicity havecaptured increasing attention due to their good biocompatibility andbiodegradability, which allows their broad biologically relevantapplications. (See, e.g., Lecomte, et al. Advances in Polymer Science,Vol. 245; 2012; pp 173-217; Engels, et al. Angew. Chem. Int. Ed. 2013,52, 9422; Datta, et al. J. Chem. Technol. Biotechnol. 2006, 81, 1119;Ikada, Y. et al. Macromol. Rapid Commun. 2000, 21, 117.) Especially, inbiomedical fields, a myriad of polyesters have been investigated withtheir potential use as drug delivery vesicles, bone screws andscaffolding or suture wire. (See, Chasin & Langer. BiodegradablePolymers as Drug Delivery Systems; Marcel Dekker Inc: New York, 1990.)Employing monomers with (strained) cyclic structures for polyestersynthesis would add stiffness to the polymer chain with substantialincrease in glass-transition temperature T_(g). (See, Van Zee et al.Angew. Chem. Int. Ed. 2015, 54, 2665; Sanford, et al. Macromolecules2016, 49, 6394; Lavilla, et al. J. Polym. Sci. A Polym. Chem. 2012, 50,1591.) The highly strained bicyclobutane-diester products from enzymaticreactions can be utilized as monomers for polyester synthesis.

Preparation of monomer dicarboxylic acids (e.g.,bicyclo[2.1.1]hexane-dicarboxylic ester). As described in Equation 12,dicarboxylic ester can be hydrolyzed under basic conditions.Dicarboxylic ester (1.0 equiv.) was treated with sodium hydroxide (5.0equiv.) in THF/H₂O (1:1 mixture, 0.1 M). The reaction was heated at 50°C. for 12 hours. The reaction mixture was then acidified with HCl (1M),and further purified by column chromatography to afford thecorresponding dicarboxylic acid. Analogously, thebicyclobutane-dicarboxylic esters can also be hydrolyzed in a similarmanner. See, e.g., Wiberg, et al. J. Am. Chem. Soc. 1983, 105, 3638

Polyester synthesis. Isoidide dicarboxylic acid (IIDCA, 1.0 equiv.) anda,w-diol (2.0 equiv.) were added into a round-bottom flask inside aKugelrohr oven. The flask was then charged with nitrogen. The oven wasthen heated to 130° C. and maintained at this temperature for 5-10 minuntil the reactants turned into a clear melt. Next, pristinedibutyltin(IV) oxide (DBTO, 1.0 mol %) was added as the catalyst intothe flask. The prepolymerization was carried out at 130° C. under acontinuous flow of nitrogen for 3 h for the polymerizations based onIIDCA. Subsequently, the polymerization temperatures were adjusted tothe desire range of 130-200° C. At the same time vacuum (0.01-0.05 mbar)was applied stepwise to the reaction system to remove the excess dioland condensates. After 3 h, the resulting polymer melt was cooled downand dissolved in chloroform and precipitated into methanol, filtered,and dried in vacuo at 30° C. for 24 h. The bicyclobutanedicarboxylicacid-based polyester could also be synthesized in this manner. (Seealso, Wu, Jet al. Macromolecules 2012, 45, 5069)

Example 4 Preparation of Bicyclobutane-Diols for Synthesis ofFunctionalized Polybenzvalene Polymers

(1R,2R)-1-(4-methoxyphenyl)bicyclo[1.1.0]butane-2,4-diyl)dimethanol wasprepared as an illustrative example, as shown in Equation 14. Diethyl(2R,4R)-1-(4-methoxyphenyl)bicyclo[1.1.0]-butane-2,4-dicarboxylate(101.3 mg, 0.33 mmol) was dissolved in anhydrous ether (10 ml) in a 50ml flask. Dry methanol (67 μl, 1.67 mmol) was added to the solution,followed by the addition of lithium borohydride (LiBH₄, 2M in THF, 0.84ml, 1.67 mmol). The reaction mixture was then heated up to a refluxingtemperature, 45° C., for 1 h, before ethyl acetate (0.5 ml) was addedand the mixture was stirred at room temperature for 30 min. Then thereaction was quenched with NH₄Cl (10 ml, sat. aq.) and diluted withwater (10 ml). The product was extracted with ethyl acetate (20 ml×6).The combined organic layer was dried over MgSO₄, and then concentratedunder reduced pressure. White solid product crashed out during theremoval of solvent. Collecting the solid product and recrystallizationwith acetone/hexane system afforded(1R,2R)-1-(4-methoxyphenyl)bicyclo[1.1.0]butane-2,4-diyl)dimethanol(52.2 mg, 0.237 mmol, 71%).

Example 5 Preparation of Multi-Substituted Cyclopropanes viaDerivatization of Cyclopropenes

Ethyl (1R,2R,3S)-3-allyl-2-methyl-2-phenethylcyclopropane-1-carboxylatewas prepared as an illustrative example. Ethyl(R)-2-phenethylcycloprop-2-ene-1-carboxylate (54.0 mg, 0.25 mmol) andCuI (5.0 mg, 0.026 mmol, 10 mol %) were dissolved in anhydrous ether (3ml) to form a suspension in a 25 ml flask. Then the flask was chargedwith argon and cooled to −78° C. Methylmagnesium bromide (0.75 ml, 1 Min ether, diluted from 3M solution in ether) was added dropwise to thereaction mixture over 10 min. The reaction was slowly warmed to −40° C.over 30 min and held at this temperature for another 1 h. A solution ofallyl bromide (43 μl, 0.50 mmol) in ether (1 ml) was then added to thereaction dropwise over 5 min. The reaction was stirred at −40° C. for 1h, before it was quenched with NH₄Cl/NH₃ (2:1, aq., 5 ml) at −20° C. Theaqueous layer was extracted twice with ether. The combined organiclayers were washed with water (10 ml), and brine (10 ml), dried overMgSO₄, and then concentrated under reduced pressure. The crude productwas purified by silica column chromatography with hexane/ethyl acetate(1:0 to 50:1 gradient) to afford ethyl(1R,2R,3S)-3-allyl-2-methyl-2-phenethylcyclopropane-1-carboxylate (48.0mg, 0.176 mmol, 71%) as a single diastereomer.

Example 6 Preparation of Multi-Substituted Fused Ring Systems viaDerivatization of Cyclopropenes

(1R,6R,7R)-3,4-Dimethyl-1-(6-((tetrahydro-2H-pyran-4-yl)methoxy)hexyl)-bicyclo[4.1.0]hept-3-ene-7-carboxylatewas prepared as an illustrative example. Ethyl(R)-2-(cyclohexylmethyl)cycloprop-2-ene-1-carboxylate (65.6 mg, 0.211mmol) was dissolved in 2,3-dimethylbutadiene (0.5 ml). Then the reactionwas heated and stirred at 80° C. in a sealed tube for 23 h. Aftercooling to room temperature, the resulting mixture was concentratedunder reduced pressure. The crude product was purified by silica columnchromatography with hexane/ethyl acetate (1:0 to 6:1 gradient) to affordethyl(1R,6R,7R)-3,4-dimethyl-1-(6-((tetrahydro-2H-pyran-4-yl)methoxy)hexyl)bicyclo[4.1.0]hept-3-ene-7-carboxylate (81.1 mg, 0.207 mmol, 98%) as asingle diastereomer.

Example 7 Click Reaction Between Terminal Alkyne-SubstitutedBicyclobutane and Azide

Diethyl(2R,4R)-1-(4-(1-((4-acetamidophenyl)sulfonyl)-1H-1,2,3-triazol-4-yl)phenyl)bicyclo[1.1.0]butane-2,4-dicarboxylate was prepared as an illustrativeexample). Diethyl(2R,4R)-1-(4-ethynylphenyl)bicyclo[1.1.0]butane-2,4-dicarboxylate (41.6mg, 0.139 mmol) was dissolved in toluene (3 ml) in a 20 ml vial.Copper(I) thiophene-2-carboxylate (CuTc, 3.2 mg, 12 mol %) was added.The mixture was kept stirring at 0° C. A suspension of4-acetamidobenzenesulfonyl azide (40.2 mg, 0.167 mmol) in toluene (5 ml)was added dropwise over 1 h. The resulting mixture was then allowed towarm to room temperature and stirred for 15 h. The reaction was dilutedwith ethyl acetate (15 ml), washed with NH₄Cl/NH₃ (2:1, 20 ml, aq.), andbrine (50 ml), dried over MgSO₄, and then concentrated under reducedpressure. The crude product was purified by silica column chromatographywith hexane/ethyl acetate (4:1 to 1 :3 gradient) to afford diethyl(2R,4R)-1-(4-(1-((4-acetamidophenyl)sulfonyl)-1H-1,2,3-triazol-4-yl)phenyl)bicyclo[1.1.0]butane-2,4-dicarboxylate (69.2 mg, 0.129 mmol, 93%).

Example 8 Identification of Biocatalysts for Strained CarbocycleFormation

General materials and methods. Unless otherwise noted, all chemicals andreagents were obtained from commercial suppliers (Sigma-Aldrich, VWR,Alfa Aesar) and used without further purification. Silica gelchromatography was carried out using AMD Silica Gel 60, 230-400 mesh. ¹Hand ¹³C NMR spectra were taken using a Bruker Prodigy 400 MHz instrumentand are internally referenced to the residual solvent peak (chloroform).Data for ¹H NMR are reported as follows: chemical shift (δ ppm),multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, p=pentet,m=multiplet, dd=doublet of doublets, dt=doublet of triplets, ddd=doubletof doublet of doublets), coupling constant (Hz), integration. Sonicationwas performed using a Qsonica Q500 sonicator. High-resolution massspectra were obtained at the California Institute of Technology MassSpectral Facility. Synthetic reactions were monitored using thin layerchromatography (Merck 60 gel plates) using a UV-lamp for visualization.Substrates were purchased from commercial suppliers.

Chromatography. Analytical reversed-phase high-performance liquidchromatography (HPLC) was carried out using an Agilent 1200 seriesinstrument and a Kromasil 100 C18 column (4.6×50 mm, 5 μm) with waterand acetonitrile as the mobile phase and visualization at 210 nm forlibrary screening. Analytical normal-phase HPLC was carried out using anAgilent 1200 series instrument and chiral columns Chiralpak IC (4.6mm×25 cm), IA (4.6 mm×25 cm), AS-H (4.6 mm×25 cm) and OJ-H (4.6 mm×25cm) with n-hexane and isopropanol as the mobile phase and visualizationat 210 nm for chiral separation. Gas chromatography (GC) analyses werecarried out using an Agilent 7820A or Shimadzu GC-17A gas chromatograph,FID detector, and a J&W HP-5 column (30 m×0.32 mm, 0.25 μm film). Gaschromatography-mass spectrometry (GC-MS) analyses were carried out usinga Shimadzu GCMS-QP2010SE system and J&W HP-5m column. Semi-preparativeHPLC was performed using an Agilent XDB-C18 column (9.4×250 mm, 5 μm)with water and acetonitrile as the mobile phase.

Cloning and site-saturation mutagenesis. pET22b(+) containing aC-terminal 6×-His tag was used as a cloning and expression vector forall enzymes described in this study. Site-saturation mutagenesis wasperformed using a modified QuikChange™ mutagenesis protocol (¹). The PCRproducts were digested with Dpnl, gel purified, and the gaps wererepaired using Gibson Mix™ (²). The ligation mixture was used todirectly transform E. coli strain BL21 E. cloni® (Lucigen). Cells weregrown using Luria-Bertani medium (LB) or Hyperbroth (AthenaES) (HB) with0.1 mg/mL ampicillin (LB_(amp) or HB_(amp)). Primer sequences areavailable upon request. Electrocompetent E. coli cells were preparedfollowing the protocol of Sambrook et al. (³). T5 exonuclease, Phusionpolymerase, and Taq ligase were purchased from New England Biolabs (NEB,Ipswich, Mass.). M9-N minimal medium (abbreviated as M9-N buffer; pH7.4) was used as a buffering system for whole cells, lysates, andpurified proteins, unless otherwise specified. M9-N buffer was usedwithout a carbon source; it contains 47.7 mM Na₂HPO₄, 22.0 mM KH₂PO₄,8.6 mM NaCl, 2.0 mM MgSO₄, and 0.1 mM CaCl₂.

Determination of hemeprotein concentration. A solution of sodiumdithionite (10 mg/mL) was prepared in M9-N buffer. Separately, asolution of 1 M NaOH (0.4 mL) was mixed with pyridine (1 mL), followedby centrifugation (10,000 g, 30 s) to separate the excess aqueous layergave a pyridine-NaOH solution. To a cuvette containing 700 μL proteinsolution (purified protein or lysate) in M9-N buffer, 50 μL ofdithionite solution (0.1 M in M9-N) and 250 μL pyridine-NaOH solutionwere added. The cuvette was sealed with Parafilm, and the UV-Visspectrum of the reduced Fe¹¹ state was recorded immediately. To anothercuvette containing 700 μL protein solution (purified protein or lysate)in M9-N buffer, 50 μL of potassium ferricyanide (0.1 M in M9-N) and 250μL pyridine-NaOH solution were added. The cuvette was sealed withParafilm, and the UV-Vis spectrum of the oxidized Fe^(III) state wasrecorded immediately. Hemeprotein concentration was determined usingε_(557[Re]-540[Ox])=23.98 mM⁻¹cm⁻¹ (⁴). Cytochrome c concentration ismeasured using a different procedure, reported previously (5).

Expression of P450 and P411 proteins. E. coli BL21 E. cloni® cellscarrying a plasmid encoding a P450 or P411 variant were grown overnightin 5 mL LB_(amp) (37° C., 250 rpm). The pre-culture was used toinoculate 45 mL of HB_(amp) in a 125 mL Erlenmeyer flask; this culturewas incubated at 37° C., 220 rpm for 2 h and 15 min. Cultures were thencooled on ice (20 min), and expression was induced with 0.5 mM IPTG and1.0 mM 5-aminolevulinic acid (final concentrations). Expression wasconducted at room temperature (23° C.), at 130 rpm, for 18-22 h.Cultures were then centrifuged (4,500 g, 5 min, 4° C.), and the pelletswere resuspended to an OD₆₀₀ of 30 in M9-N minimal medium (no nitrogen).Aliquots of the cell suspension (4 mL) were used to determine the P450and P411 expression level after lysis by sonication. The expressionlevel in OD₆₀₀=30 lysates is typically in the range 8-12 μM for P450s,1-4 μM for P450 H*, and 2-7 μM for P411s.

The same procedure was used for expression of globin proteins, exceptusing 1 mL LB_(amp) pre-culture to inoculate 49 mL of HB_(amp). Theexpression of cytochrome c variants requires an extra maturation plasmidpEC₈₆ in E. coli BL21 E. cloni® cells and follows a procedure describedpreviously (⁵). Biotransformations. The cell suspension in M9-N (with acertain OD₆₀₀) was degassed by sparging with argon in sealed vials orflasks for 30 min. Separately, a glucose solution (250 mM in M9-N) wasdegassed by sparging with argon for 30 minutes. All solutions wereuncapped and transferred into an anaerobic chamber. Resuspended cells(340 μL) were added to 2 mL vials, followed by glucose (40 μL, 250 mM inM9-N), alkyne (10 μL of an EtOH stock), and EDA (10 μL of an EtOHstock). Final concentrations were typically 10.0-20.0 mM alkyne,10.0-20.0 mM EDA (alkyne: EDA=1:1), and 25 mM glucose; final reactionvolume was 400 μL. The vials were sealed, removed from the anaerobicchamber, and shaken at room temperature and 560 rpm for 6 h. After thereaction was completed, internal standard 1,3,5-trimethoxybenzene (20 μLof 20 mM stock solution in toluene) was added to the reaction vialfollowed by mixed solvent (cyclohexane/ethyl acetate=1:1, 1.0 mL). Themixture was transferred to a 1.5 mL microcentrifuge tube, and thensubjected to vortex (15 s×3) and centrifugation (14,000 rpm, 5 min) tocompletely separate the organic and aqueous layers. A sample of theorganic layer (0.8 mL) was transferred to a vial for GC analysis. Theprocedure for preparative-scale enzymatic reactions is outlined indetail (See Section VIII and IX).

Reaction screening in 96-well plate format. Single-site-saturationlibraries were generated employing the “22c-trick” method (l) andscreened in one 96-well plate; double-site-saturation libraries weregenerated using the same method to target two different sites; thesewere screened in three 96-well plates. E. coli libraries were culturedin LB_(amp) (300 μL/well) at 37° C., 250 rpm and 80% relative humidityovernight. HB_(amp) (950 μL/well) was inoculated with the pre-culture(50 μL/well) and incubated at 37° C., 250 rpm, 80% humidity for 2.5 h.The plates were cooled on ice for 30 minutes, and expression was inducedwith 0.5 mM IPTG and 1.0 mM 5-aminolevulinic acid (finalconcentrations). Expression was conducted at 20° C. and 200 rpm for18-22 h. The cells were pelleted (4,500 g, 5 min, 4° C.) and resuspendedwith M9-N buffer (340 μL/well) and D-glucose solution (40 μL/well, inM9-N). The 96-well plate was then transferred to an anaerobic chamber.In the anaerobic chamber, alkyne (10 μL/well, 400 mM in EtOH) and EDA(10 μL/well, 400 mM in EtOH). The plate was sealed with an aluminumfoil, removed from the anaerobic chamber, and shaken at 560 rpm. Seebelow for reaction workup protocol:

Bicyclobutane formation screening. After 16 h, the seal was removed andmixed solvent (cyclohexane/ethyl acetate=1:1, 600 μL/well) and internalstandard 1,3,5-trimethoxybenzene (20 μL/well of a toluene stock) wereadded. The plate was tightly sealed with a reusable silicone mat,vortexed (15 s×3) and centrifuged (4,500 g, 5 min) to completelyseparate the organic and aqueous layers. The organic layers (200μL/well) were transferred to 300 μL vial inserts, which were then placedin 2 mL vials labeled with corresponding wells in the plate for GC orGC-MS analysis (a 2.5-min/run method was used for screening, see SectionIV).

Cyclopropene formation screening. After 16 h, the seal was removed andacetonitrile (580 μL/well) and internal standard ethyl phenylacetate(PhEA, 20 mM in acetonitrile, 20 μL/well) were added. The plate wastightly sealed with a reusable silicone mat, vortexed (15 s×3) andcentrifuged (4,500 g, 5 min). The supernatant (200 μL/well) was filteredthrough an AcroPrep 96-well filter plate (0.2 μm) into a shallow-wellplate for HPLC analysis (a 1.7-min/run method was used for screening,see Section V).

General procedure for testing transition metal catalysts for designedcarbene transfer to alkyne. To an 8 mL vial were added phenyl acetylene(0.1 mmol), catalyst (Rh-1 mol %; Cu-10 mol %) and DCM or cyclohexane (2mL). The vial was cooled to −78° C. EDA (0.2 mmol in 0.5 mLcorresponding organic solvent) was added through a syringe pump over 1h. The reaction was stirred at −78° C. for 6 h and then slowly warmed toroom temperature over another 6 h. The reaction mixture (0.5 mL) wasfiltered through a 2 cm-height silica gel and washed with DCM (1 mL).The filtrate was used for GC-MS analysis.

The procedure for testing hemeproteins follows that described aboveusing E. coli expressing corresponding hemeprotein variant as whole-cellcatalyst (OD₆₀₀=30). For testing heme (±BSA), 0.5 mM stock of heme(±BSA, 0.75 mg/mL) in M9-N (340 μL) and Na₂S₂O₄ (40 μL, 10 mM in M9-N)was used instead of cell suspension and glucose stock. (BSA=bovine serumalbumin).

2-Ethoxy-5-phenylfuran (3b). ¹H NMR (400 MHz, Benzene-d₆) δ 7.58 (dd,J=8.3, 1.3 Hz, 2H), 7.14-7.09 (m, 2H), 6.97 (tt, J=7.4, 1.3 Hz, 1H),6.37 (d, J=3.3 Hz, 1H), 5.02 (d, J=3.3 Hz, 1H), 3.59 (q, J=7.0 Hz, 2H),0.98 (t, J=7.0 Hz, 3H). ¹³C NMR (101 MHz, C₆D₆) δ 161.79, 145.08,132.15, 129.52, 127.04, 123.49, 107.28, 83.71, 67.27, 15.07. MS (EI)m/z: 188 (M^(+·)); calc. for C₁₂H₁₂O₂: 188.

Ethyl 2-phenyl-3-(trimethylsilyl)cycloprop-2-ene-1-carboxylate (3c). Thesynthesis of 3c follows a reported procedure (⁶). ¹H NMR (400 MHz,CDCl₃) δ 7.61-7.52 (m, 2H), 7.48-7.36 (m, 3H), 4.25-4.06 (m, 2H), 2.40(s, 1H), 1.25 (t, J=7.1 Hz, 3H), 0.34 (s, 9H). ¹³C NMR (101 MHz, CDCl₃)δ 176.13, 129.79, 129.52, 128.65, 127.56, 123.95, 108.00, 60.00, 20.60,14.37, −1.43. MS (EI) m/z: 260 (NT^(+·)); calc. for C₁₅H₂₀O₂Si: 260.

Ethyl 2-phenylcycloprop-2-ene-1-carboxylate (3a). To a 20 mL vial wereadded 3c (20.8 mg, 0.08 mmol) and THF (2 mL). And thentetrabutylammonium fluoride (TBAF, 1M in THF, 120 μL) was added to thesolution. The reaction was stirred at room temperature for 3 min beforeit was condensed under reduced pressure (3 min). Quick purification(within 5 min) of the crude product by silica column chromatography withhexane/ethyl acetate (10:1) afforded 3a. 3a was used for qualitativeGC-MS analysis. (Note: 3a is highly unstable. It was completelydecomposed under deprotection condition after 30 min. Purification stepneeds to be very quick. After column purification, it is better to leave3a in the hexane/ethyl acetate eluent rather than concentrate it underreduced pressure. 3a can stay for hours in a solution form, but for lessthan 30 min in pure form.) MS (EI) m/z: 188 (M^(+·)); calc. forC₁₂H₁₂O₂: 188.

Protein purification. E. coli BL21 E. cloni R cells carrying a plasmidencoding a P411 variant were grown overnight in 105 mL LB_(amp) (37° C.,250 rpm). HB_(amp) (1 L) in a 2.8 L flask was inoculated with 100 mL ofthe pre-culture and incubated at 37° C. and 240 rpm for 2 h and 15 min.Cultures were then cooled on ice (30 min) and induced with 0.5 mM IPTGand 1.0 mM 5-aminolevulinic acid (final concentrations). Expression wasconducted at 20° C., 130 rpm, for 20 h. Cultures were then centrifuged(4,500 g, 8 min, 4° C.) and the cell pellets frozen at 20° C. Forpurification, frozen cells from two such cultures were resuspended inbuffer A (25 mM Tris-HCl buffer, 20 mM imidazole, 100 mM NaCl, pH 7.5, 4mL/g of cell wet weight), loaded with hemin (1 mg/gram wet cell weight)and powdered DNasel, and lysed by sonication. To pellet cell debris,lysates were centrifuged (20,000×g, 20 min, 4° C.). Proteins wereexpressed in a construct containing a 6×-His tag and purified using anickel NTA column (1 mL HisTrap HP, GE Healthcare, Piscataway, N.J.)using an AKTA or AKTAxpress purifier FPLC system (GE healthcare). P411enzymes were eluted with a linear gradient from 100% buffer A to 100%buffer B (25 mM tris, 300 mM imidazole, 100 mM NaCl, pH 7.5) over 10column volumes. Proteins were then pooled, concentrated, and subjectedto three exchanges of phosphate buffer (0.1 M KPi, pH 8.0) usingcentrifugal filters (10 kDa molecular weight cut-off, Amicon Ultra,Merck Millipore) to remove excess salt and imidazole. Concentratedproteins were aliquoted, flash frozen on powdered dry ice, and stored at−80 or −20° C.

Results and discussion. In cyclic organic molecules, ring strain arisesfrom distortions of bond angle and bond length, steric clashes ofnon-bonded substituents, and other effects (⁷). The simplestcarbocycles, cyclopropanes and cyclobutanes, possess ring strains of26-28 kcal/mol (⁸). Introducing carboncarbon multiple bonds or bridgesto these small ring systems induces additional strain as well asstructural rigidity. For example, cyclopropenes with an endo-cyclicdouble bond bear a strain of 54 kcal/mol, whereas bicyclo[1.1.0]butanes,folded into puckered structures, distinguish themselves as one of themost strained four-membered systems, with around 66 kcal/mol strain(FIG. 4) (8). These carbocycles are particularly attractiveintermediates in chemical and materials synthesis, since they canundergo strain-release transformations to furnish a myriad of usefulscaffolds (⁹-¹²). The structural rigidity imparted by strained rings insupramolecular materials can lead to interesting physical properties,such as mechanical stability (¹³) and high glass-transition temperature(¹⁴). The intrinsic energy of these strained structures can also berelieved in response to exogenous force, which leads to radical changesin physical properties (e.g., conductivity), a feature highly desirablefor stimulus-responsive materials (¹⁵, ¹⁶).

High ring strain, however, greatly increases the difficulty ofsynthesis. A commonly used method for preparing bicyclobutanes startsfrom dibromo-2-(bromomethyl)cyclopropane substructures and utilizesorganolithium reagents for lithium-halogen exchange followed bynucleophilic substitution under rigorously anhydrous and cryogenicconditions (9). An alternative route relies on the double transfer of acarbene to alkynes, but the few examples in the literature are mostlylimited to methylene carbene (¹⁷-¹⁹). Asymmetric bicyclobutaneconstruction is particularly challenging, with multiple chiral centersgenerated at the same time (²⁰, ²¹) (FIG. 5). Cyclopropene synthesisthrough enantioselective single carbene addition to alkynes alsorequires chiral transition metal catalysts based on rhodium (²², ²³))iridium (²⁴) and cobalt (²⁵). Development of a sustainable catalyticsystem that performs with high efficiency and selectivity under ambientconditions would be a significant advance for construction of theseuseful, highly strained carbocycles.

Enzymes, the catalytic workhorses of biology, are capable ofaccelerating chemical transformations by orders of magnitude whileexhibiting exquisite control over selectivity (²⁶). Although naturesynthesizes various cyclopropane-containing products (²⁷), cyclopropeneor bicyclobutane fragments are extremely rare (FIG. 6)(²⁸, ²⁹). This maybe attributed to the lack of biological machinery for synthesizing thesemotifs and/or the instability of these structures under biological ornatural product isolation/purification conditions. Nevertheless, thestudies disclosed herein were conducted upon envisioning that existingenzymes could be repurposed to forge strained carbocycles by takingadvantage of their catalytic promiscuity (³⁰, ³¹) in the presence ofnon-natural substrates and by using directed evolution to optimize theactivity and selectivity of these starting enzymes (³²).

In the past several years, the present inventors and others haveengineered natural hemeproteins to catalyze reactions not known innature (³³-³⁸). The present studies were begun with the hypothesis thatcarbene transfer to triple bonds with a heme-dependent enzyme mightafford highly strained cyclopropene and bicyclobutane structures and doso enantioselectively. Several challenges were expected at the outset,especially in chiral bicyclobutane formation, as it involves twosequential carbene additions to the alkyne substrate: 1) the enzymewould need to bind the alkyne in a specific conformation in order totransfer the carbene enantioselectively; 2) the high-energy cyclopropeneintermediate generated by the first carbene addition would need to beaccepted and stabilized by the protein; 3) compared to methylene carbeneused previously, a substituted carbene (e.g., with an ester group) mighthinder access of the cyclopropene to the iron-carbenoid; and 4) theprotein would also need to exert precise stereocontrol over the secondcarbene transfer step regardless of structural differences between theinitial alkyne and the cyclopropene intermediate. Despite thesechallenges, an investigation was initiated to determine whether astarting enzyme with this unusual and non-natural activity could beidentified, and whether its active site could be engineered to create asuitable environment for substrate binding, intermediate stabilization,and selective product formation.

First, free heme (±bovine serum albumin (BSA)), which is known tocatalyze styrene cyclopropanation (33), was tested to see if it couldtransfer carbenes to alkynes. Reactions using ethyl diazoacetate (EDA)and phenylacetylene (1a) as substrates in neutral buffer (M9-N minimalmedium, pH 7.4) at room temperature, however, gave no cyclopropene orbicyclobutane product. Next, a panel of hemeproteins includingcytochromes P450, cytochromes P411 (P450 with the axial cysteine ligandreplaced by serine), cytochromes c and globins in the form of E. coliwhole-cell catalysts were tested for the desired transformation underanaerobic conditions (38), but none were fruitful (FIG. 7 and Table 2).

TABLE 2 Screening different catalysts for bicyclobutane formation

Ineffective transition metal catalysts (with undefined productmixtures): Rh₂(OAc)₄ Rh₂(OPiv)₄ Rh₂(S-DOSP)₄ Rh₂(esp)₂ CuI CuOTf•PhHCuCl•NHC Cu(OTf)₂ Ineffective heme or hemeprotein catalysts (with noproduct observed): heme heme + BSA P450-WT P450-CIS P411-CIS P450-CIS H*Rma cyt c Rma cyt c TDE myoglobin myoglobin VA Rma NOD Q52V Ape pgb Y60GEffective hemeprotein catalysts (with product detected): P411-Si I263W:210 ± 20 TTN, 3b only P411-Si I263F: ~10 UN, 3b; <5 TTN, 2a P411-Si1263F A328V (P2): 23 ± 8 TTN, 2a: 3b ~10:1 P411-S1 V87A I263F A328V(P3): 60 ± 10 TTN, 2a: 3b >20:1 P411-S1 V87A I263F A268G A328V (P4): 80± 10 TTN, 2a: 3b >50:1 P411-P4 A821: 280 ± 30 UN, 2a: 3b >50:1 P411-P4A78V A82L F263L (E10): 530 ± 20 TTN, 2a: 3b >50:1(Abbreviations: Piv=pivaloyl, DOSP=N-(p-dodecylphenylsulfonyl)prolinato,esp=α,α,α′,α′-tetramethyl-1,3-benzenedipropionic acid,Tf=trifluoromethanesulfonyl,NHC=1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene.)

TABLE 3 Information of hemeprotein variants Name Description MutationsP450-WT P450_(BM3)-wild type — (CYP102A1) (³⁹) P450-CIS P450_(BM3)-CISvariant (³³) V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G,T268A, A290V, L353V, I366V, E442K (relative to P450-WT) P411-CISP411_(BM3)-CIS variant (³⁸) C400S (relative to P450-CIS) P450-CIS H*P450_(BM3)-CIS H* variant (⁴⁰) C400H (relative to P450-CIS) P411-S1P411_(BM3)-CIS S1 variant (³⁸) T438S (relative to P411-CIS) P411-“I263F”P411_(BM3)-CIS “I263F“ variant (⁴¹) I263F (relative to P411-S1) P411-P2P411_(BM3)-CIS P2 variant (³⁴) I263F, A328V (relative to P411-S1)P411-P3 P411_(BM3)-CIS P3 variant (³⁴) V87A, I263F, A328V (relative toP411-S1) P411-P4 P411_(BM3)-CIS P4 variant (³⁴) V87A, I263F, A268G,A328V (relative to P411-S1) P411-“A82L” P411_(BM3)-CIS “A82L” variant⁽³⁴) A82L, V87A, I263F, A268G, A328V (relative to P411-S1) P411-E10P411_(BM3)-CIS E10) variant (³⁵) A78V, A82L, V87A, I263L, A268G, A328V(relative to P411- S1) Rma cyt c Rhodothermus marinus — cytochrome cwild-type (⁵) Rma cyt c Rhodothermus marinus V75T, M100D, M103E(relative to TDE cytochrome c TDE variant (⁵) Rma cyt c) myoglobin Spermwhale myoglobin-wild — type (⁴²) myoglobin Sperm whale myoglobin-VAH64V, V68A (relative to VA variant (Error! Bookmark not Myoglobin)defined.) Rma NOD Rhodothermus marinus nitric Q52V (relative to RmaNOD-wild Q52V oxide dioxygenase Q52V variant type) Ape pgb Aeropyrumpernix protoglobin Y60G (relative to Ape pgb wild- Y60G Y60G varianttype)

Interestingly, a P411 variant obtained in a previous cyclopropanationstudy, P411-S1 1263W (see Supplementary Materials for sources, sequencesand mutations), afforded a furan product (3b) with a total turnovernumber (TTN) of 210. Other furan analogs have been identified as adductsof carbenes and alkynes (⁴³), which prompted the consideration of howfuran 3b was generated. Preliminary kinetic study of the enzymaticreaction suggested that the enzyme first synthesized an unstablecyclopropene (3a), which subsequently rearranged to the furan eitherspontaneously or with assistance from the enzyme (FIG. 7B and FIG. 8).For the time-course study, reactions were set up in parallel accordingto the procedure in biotransformations using whole-cell catalyst(OD₆₀₀=30). The reactions were quenched at certain time points andanalyzed by GC-MS. Every data point (average of two reaction) in FIG. 8is the peak area of cyclopropene 3a or furan 3b divided by that ofinternal standard on GC-MS. Cyene=cyclopropene. These results providedstrong evidence that the P411 hemeprotein is capable of transferring acarbene to an alkyne, which is an activity not previously reported forany protein or even any iron complex.

Example 9 Engineering of Biocatalysts for Bicyclobutane Formation

To divert the enzymatic reaction to bicyclobutane formation, the enzymewould have to transfer a second carbene to cyclopropene intermediate 3abefore the cyclopropene rearranges to the undesired furan product (FIG.7B). Therefore, P411 variants closely related to P411-S1 1263W weretested. It was reasoned that amino acid residue 263, which resides inthe distal pocket, above the heme cofactor, might modulate the rate ofthis step and that the bulky tryptophan (Trp) side chain at this sitemay be blocking the second carbene transfer. A P411-S1 variant withphenylalanine (Phe) instead of Trp at this position (I263F) in factcatalyzed bicyclobutane formation at a very low level (<5 TTN) (Table2). Variant ‘P4’ with 3 additional mutations relative to P411-S1 I263F(V87A, A268G and A328V) (34) synthesized the desired bicyclobutane 2awith 80 TTN and with the formation of furan adduct substantiallysuppressed (2a:3b>50:1, FIG. 7C).

Another related P411 variant, E10 (=P4 A78V A82L F263L), which wasengineered from P4 for nitrene transfer reactions (35), catalyzed thedesired transformation with >6-fold higher activity (530 TTN, FIG. 7E).NMR analysis revealed an exo, endo-configuration of theenzymatically-produced bicyclobutane 2a, which is distinct from the onlyreported achiral endo, endo-isomer, made using an osmium-porphyrincomplex (⁴⁴, ⁴⁵).This P411-E10 variant was chosen as the startingtemplate for directed evolution of an even more efficientbicyclobutane-constructing enzyme.

Because the side chain of residue 263 influenced formation of thebicyclobutane product, site-saturation mutagenesis (SSM) of variant E10was performed at position 263 and screened whole E. coli cellsexpressing the mutated proteins for improved production of bicyclobutane2a. The enzyme having leucine at this position (263L) was the mostactive; other amino acid residues either lowered the reactivity towardsbicyclobutane formation and/or delivered more furan product. Inparallel, two additional residues in E10, V78 and S438, were alsotargeted by SSM. Aromatic residues were found to be activating at 78,with a phenylalanine or tyrosine mutation giving 1.5-2-fold improvementover E10. This beneficial effect may stem from a 7C-7C stackinginteraction between the side chain and the alkyne substrate or thecyclopropene intermediate. A single S438A mutation on a loop residingabove the heme also significantly increased the activity,giving >2.5-fold increase in turnover. Finally, recombination of V78F/Yand S438A mutations led to the discovery of even more powerfulbiocatalysts for bicyclobutane formation (e.g., 1880 TTN with E10 V78FS438A, FIG. 7E and FIG. 9).

Data in FIG. 9 are also summarized in Table 4. Analytical reactions wereset up in quadruplicate according to the procedure forbiotransformations described above using whole-cell catalyst andanalyzed by GC using the standard calibration curve of 2a. The TTNs wereobtained for OD₆₀₀=30 for P4, A82L and E10 variants, OD₆₀₀=15 for E10V78Y and E10 V78F variants, and OD₆₀₀=10 for E10 S438A, E10 V78Y S438Aand E10 V78F S438A variants. TTNs reported are the average of biologicalduplicates. Each biological set contains four experiments. The errors inall tables are standard deviations.

TABLE 4 P411-S1 variants for bicyclobutane formation P411-S1 variantsTTN P4  80 ± 10 “A82L”  280 ± 20 E10  530 ± 20 E10 V78Y  800 ± 30 E10V78F 1030 ± 60 E10 S438A 1400 ± 50 E10 V78Y S438A 1560 ± 70 E10 V78FS438A 1880 ± 80

TABLE 5 Mutations in related P411-E10 variants Name DescriptionMutations relative to P450-WT P4 P411_(BM3)-CIS P4 V78A, F87A, P142S,T175I, variant (³⁴) A184V, S226R, H236Q, E252G, I263F, T268G, A290V,A328V, L353V, I366V, C400S, T438S, E442K “A82L” P411_(BM3)-CIS “A82L”V78A, A82L, F87A, P142S, variant (³⁴) T175I, A184V, S226R, H236Q, E252G,I263F, T268G, A290V, A328V, L353V, I366V, C400S, T438S, E442K E10P411_(BM3)-CIS E10 A82L, F87A, P142S, T175I, variant (³⁵) A184V, S226R,H236Q, E252G, I263L, T268G, A290V, A328V, L353V, I366V, C400S, T438S,E442K E10 V78Y P411_(BM3)-CIS E10 V78Y V78Y, A82L, F87A, P142S, T175I,A184V, S226R, H236Q, E252G, I263L, T268G, A290V, A328V, L353V, I366V,C400S, T438S, E442K E10 V78F P411_(BM3)-CIS E10 V78F V78F, A82L, F87A,P142S, T175I, A184V, S226R, H236Q, E252G, I263L, T268G, A290V, A328V,L353V, I366V, C400S, T438S, E442K E10 S438A P411_(BM3)-CIS E10 S438AA82L, F87A, P142S, T175I, A184V, S226R, H236Q, E252G, I263L, T268G,A290V, A328V, L353V, I366V, C400S, T438A, E442K E10 V78Y P411_(BM3)-CISE10 V78Y V78Y, A82L, F87A, P142S, S438A S438A T175I, A184V, S226R,H236Q, E252G, I263L, T268G, A290V, A328V, L353V, I366V, C400S, T438A,E442K E10 V78F P411_(BM3)-CIS E10 V78F V78F, A82L, F87A, P142S, S438AS438A T175I, A184V, S226R, H236Q, E252G, I263L, T268G, A290V, A328V,L353V, I366V, C400S, T438A, E442K

Example 10 Analysis of Alkyne Coupling Partners

With the evolved E10 V78F S438A variant in hand, the bacterial catalystwas then assayed against a panel of aromatic alkyne coupling partners.Biotransformations with 10 different substrates were performed on0.1-0.2 mmol scale. These preparative-scale reactions proceeded smoothlyto furnish the corresponding bicyclobutanes with up to 1760 TTN and 80%yield (FIG. 11A). Additionally, three alkynes, 1k, 1l and 1m, weretransformed in mmol scale, and bicyclobutanes were isolated inhundred-milligram quantities, demonstrating that the biocatalytictransformation is readily scalable. Among the 13 different substrates,the engineered P411 hemeprotein did not exhibit strong preference towardspecific electronic or steric features. Electron-deficient halides(2b-2d), which can be used as pre-functionalities for furthertransformations as well as electron-rich alkyl or alkoxy groups (2e-2hand 2k) at meta- or para-position of the phenyl group were accepted bythe enzyme. Even heterocyclic substrates such as thiophene (2j) servedas suitable alkyne partners, albeit with lower reactivity.

Free functionalities, including alcohols (2i and 2m) and a second alkyne(2l), are well-preserved, providing an additional opportunity forderivatization of these products. A terminal alkyne allowscopper-catalyzed click chemistry, through which bicyclobutane 2l can bemodified with a simple sulfonyl azide (4a) or even decorated withbiologically relevant fragments, such as a phenylalanine derivative(4b). An unprotected hydroxyl group could also offer the possibility oflinkage to useful structures. Additionally, in order to probe theenantiopurity of bicyclobutane products, 2l and 2m were derivatized withL-azido-phenylalanine and (R)-Mosher's acid, respectively. Thediastereomeric excess of these derivatized products would indicate theenantiomeric ratio of the bicyclobutanes. In fact, only singlediastereomers of derivatized bicyclobutanes 4b and 4c were observed byNMR. Furthermore, the dicarboxylic esters on the bicyclobutane structurecan be reduced easily with a mild reducing reagent, LiBH₄, to give diolproduct 4d with the strained ring structure preserved. The diol product4d allowed for the unequivocal confirmation of the bicyclobutanestructure and determination of the absolute configuration through X-raycrystallography.

Example 11 Analytical-Scale and Preparative-Scale EnzymaticBicyclobutane Formation

All enzymatic reactions for bicyclobutane formation in analytical scalewere conducted following the general procedure described below andanalyzed with gas chromatography (GC). All TTNs for the differentproducts were approximated using the GC standard curve of bicyclobutane2a, assuming substituents on the phenyl ring will not significantlyaffect the GC signals. These estimated TTNs were only used to helpdetermine the reaction scale required to obtain sufficient bicyclobutaneproducts for isolation, purification and characterization.

General procedure for analytical-scale reactions. To a 2 mL vial wereadded degassed suspension of E. coli expressing P411-E10 V78F S438Avariant in M9-N buffer (OD₆₀₀=9, 340 μL), alkyne (10 μL of 200 mM stocksolution in EtOH, 5 mM final concentration), EDA (10 μL of 400 mM stocksolution in EtOH, 10 mM final concentration, 2.0 equiv.), D-glucose (40μL of 250 mM stock solution in M9-N buffer, 25 mM final concentration)under anaerobic conditions. The vial was capped and shaken at 560 rpm atroom temperature for 6 h. After the reaction was completed, internalstandard 1,3,5-trimethoxybenzene (20 μL of 20 mM stock solution intoluene) was added to the reaction vial followed by mixed solvent(cyclohexane/ethyl acetate=1:1, 1 mL). The mixture was transferred to a1.5 mL microcentrifuge tube, and then vortexed (15 s×3) and centrifuged(14,000 rpm, 5 min) to completely separate the organic and aqueouslayers. 0.8 mL of organic layer was taken for GC analysis. TTN wascalculated based on measured protein concentration. Reactions for everysubstrate were set up in quadruplicate.

During testing of analytical reactions, electron-withdrawing alkynes 1nand 1o also gave fair amount of products, which were detected by GC-MS.However, problems in isolating these products from preparative-scalereactions were encountered. These results are noteworthy, but theycannot be used for drawing conclusions before further validation. Theresults of the analytical reactions are shown in Table 6.

TABLE 6 Analysis data for analytical-scale bicyclobutane formationreactions [Pdt]^(c)/ [PC]^(d)/ Avg. Avg. Entry Pdt^(a) Std^(b) Pdt/StdmM μM TTN TTN yield 2a-(1) 3012.4 593.0 5.080 3.12 1.66 1882 2a-(2)3010.1 580.6 5.184 3.18 1.66 1921 2a-(3) 2851.0 585.8 4.867 2.98 1.661803 2a-(4) 2921.9 565.9 5.163 3.17 1.66 1913 1880 62.2 2b-(1) 3204.9554.0 5.785 3.47 1.66 2097 2b-(2) 3278.8 558.0 5.876 3.53 1.66 21302b-(3) 3274.2 565.1 5.794 3.48 1.66 2100 2b-(4) 3154.2 559.6 5.637 3.381.66 2043 2092 69.3 2c-(1) 2479.6 549.9 4.509 2.71 1.66 1634 2c-(2)2406.8 577.9 4.165 2.50 1.66 1510 2c-(3) 2317.3 577.5 4.013 2.41 1.661455 2c-(4) 2185.1 587.0 3.722 2.23 1.66 1349 1438 49.2 2d-(1) 2007.2576.5 3.482 2.09 1.66 1262 2d-(2) 1673.5 564.7 2.964 1.78 1.66 10742d-(3) 1436.5 570.1 2.520 1.51 1.66 913 2d-(4) 1572.0 559.6 2.809 1.691.66 1018 1067 35.3 2e-(1) 2712.6 551.2 4.921 2.95 1.66 1784 2e-(2)2943.0 575.5 5.114 3.07 1.66 1854 2e-(3) 2565.4 556.1 4.613 2.77 1.661672 2e-(4) 2725.3 558.7 4.878 2.93 1.66 1768 1769 58.6 2f-(1) 1265.4582.8 2.171 1.30 1.66 787 2f-(2) 1261.3 583.0 2.163 1.30 1.66 784 2f-(3)1247.4 574.0 2.173 1.30 1.66 788 2f-(4) 1194.4 580.7 2.057 1.23 1.66 746773 25.7 2g-(1) 2899.6 605.2 4.791 2.87 1.66 1737 2g-(2) 2881.5 587.14.908 2.94 1.66 1779 2g-(3) 2797.3 586.1 4.773 2.86 1.66 1730 2g-(4)2657.8 571.2 4.653 2.79 1.66 1687 1733 57.4 2j-(1) 244.5 605.1 0.4040.24 1.66 147 2j-(2) 255.0 625.4 0.408 0.24 1.66 148 2j-(3) 256.9 618.40.415 0.25 1.66 151 2j-(4) 231.0 633.1 0.365 0.22 1.66 132 145 4.82k-(1) 2837.1 565.1 5.021 3.01 1.66 1820 2k-(2) 2818.8 591.5 4.766 2.861.66 1727 2k-(3) 2792.4 572.9 4.874 2.92 1.66 1767 2k-(4) 2824.5 581.34.859 2.92 1.66 1761 1769 58.6 2l-(1) 1921.1 566.3 3.392 2.04 1.66 12302l-(2) 1703.9 549.4 3.101 1.86 1.66 1124 2l-(3) 1797.8 565.5 3.179 1.911.66 1152 2l-(4) 1656.7 565.1 2.932 1.76 1.66 1063 1113 37.8 ^(a)Pdt =product area on GC, ^(b)Std = internal standard area on GC, ^(c)[Pdt] =product concentration in reaction mixture, ^(d)[PC] = proteinconcentration in initial reaction mixture, Avg. = average.

All enzymatic reactions for bicyclobutane formation in preparative scalewere conducted following the general procedure described below and thecorresponding bicyclobutane products were isolated. Detailed conditionsfor the preparative-scale reactions of different substrates areindicated separately.

General procedure for preparative-scale reactions. To a 40 mL vial or250 mL flask were added degassed suspension of E. coli expressing P411E10-V78F S438A variant (OD₆₀₀=10-20), alkyne (0.1-0.2 mmol, largerscales for 1k-1m), EDA (2.0-4.0 equiv.), D-glucose (10-15 mM, 250 mMstock in M9-N), 1-5 vol % EtOH, M9-N buffer (pH 7.4) under anaerobicconditions. The vial or flask was capped and shaken (420 rpm for vialsand 220 rpm for flasks) at room temperature for 12 h.

After the reaction was completed, every 30 mL portion ofpreparative-scale reaction mixture was transferred to a 50 mL Falconcentrifuge tube. The reaction container was washed with water (2 mL×2)followed by mixed organic solvent (cyclohexane/ethyl acetate =1:1, 2mL×3). The washing solution was combined with the reaction mixture inthe centrifuge tubes. Additional 12 mL of cyclohexane/ethyl acetatesolvent was added to every tube. After the tube (with 45 mL mixture intotal) was capped, it was vortexed (1 min×3), shaken vigorously, andcentrifuged (14,000 g, 5 min). The organic layer was separated and theaqueous layer was subjected to three more rounds of extraction. Thenorganic layers were combined, dried over Na₂SO₄, and concentrated underreduced pressure. Purification by silica column chromatography withhexane/ethyl acetate as eluent afforded the desired bicyclobutanes. TTNswere calculated based on measured protein concentration and the isolatedyield of the product.

Diethyl (2R,4R)-1-phenylbicyclo[1.1.0]butane-2,4-dicarboxylate (2a)

E. coli suspension in M9-N D-glucose in M9-N volume/ n_pro/ volume/[Glu]/ OD₆₀₀ mL [PC]/μM μmol mL mM 20 24.0 4.17 0.100 1.0 ~10 alkyne(1a) stock in EtOH EDA stock in EtOH stock/M volume/μL n_1/mmol stock/Mvolume/μL n_2/mmol 1.00 180 0.18 2.00 180 0.36 product purificationeluent m[Pdt]/mg n Pdt]/mmol yield TTN Ethyl acetate in 31.3 0.114 63%1140 hexanes (0% to 10% gradient)

In the tables for compounds 2a-2m, [PC]=protein concentration inoriginal cell suspension, n_pro=amount of protein in the reaction,[Glu]=D-glucose concentration in reaction mixture, n_1=amount of alkynein the reaction, n_2 =amount of EDA in the reaction, m[Pdt]=mass ofproduct isolated, n[Pdt]=amount of product.

¹H NMR (400 MHz, CDCl₃) δ 7.34-7.22 (m, 5H), 4.14 (qd, J=7.2, 2.4 Hz,2H), 4.07 (qd, J=7.2, 3.7 Hz, 2H), 3.26 (s, 1H), 3.13 (d, J=3.0 Hz, 1H),3.10 (d, J=3.0 Hz, 1H), 1.27 (t, J=7.1 Hz, 3H), 1.13 (t, J=7.1 Hz, 3H).¹³C NMR (101 MHz, CDCl₃) δ 170.36, 167.91, 132.68, 128.50, 128.45,127.56, 60.99, 60.91, 44.64, 42.55, 28.18, 20.47, 14.37, 14.16. HRMS(FAB) m/z: 275.1271 (M+H⁺); calc. for C₁₆H₁₉O₄: 275.1283. [α]²³_(D)=124.8±1.1° (c 0.1, ethyl acetate).

Diethyl (2R,4R)-1-(4-fluorophenyl)bicyclo[1.1.0]butane-2,4-dicarboxylate(2b)

E. coli suspension in M9-N D-glucose in M9-N volume/ [PC]/ n_pro/volume/ [Glu]/ OD₆₀₀ mL μM μmol mL mM 20 24.0 4.17 0.100 1.0 ~10 alkyne(1b) stock in EtOH EDA stock in EtOH volume/ n_1/ volume/ n_2/ stock/MμL mmol stock/M μL mmol 1.0 180 0.18 2.0 180 0.36 purification eluentproduct Ethyl acetate m[Pdt]/ n[Pdt]/ in hexanes mg mmol yield TTN (0%to 10% gradient) 36.6 0.125 70% 1250

¹H NMR (400 MHz, CDCl₃) δ 7.32-7.26 (m, 2H), 7.05-6.94 (m, 2H),4.21-4.11 (m, 2H), 4.11-4.00 (m, 2H), 3.23 (d, J=0.5 Hz, 1H), 3.09 (d,J=3.0 Hz, 1H), 3.04 (dd, J=3.0, 0.6 Hz, 1H), 1.27 (t, J=7.1 Hz, 3H),1.14 (t, J=7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 170.20, 167.80,163.59 (d, Jc-F =246.4 Hz), 130.55 (d, Jc-F =8.2 Hz), 128.31, (d, Jc-F=3.3 Hz), 115.63, (d, J_(C-F)=21.8 Hz), 61.06, 60.98, 44.83, 42.70,27.50, 19.89, 14.37, 14.20. HRMS (FAB) m/z: 293.1179 (M+H⁺); calc. forC₁₆H₁₈FO₄: 293.1189. [α]²³ _(D)=−110.1±2.0° (c 0.1, ethyl acetate).

Diethyl (2R,4R)-1-(4-chlorophenyl)bicyclo[1.1.0]butane-2,4-dicarboxylate(2c)

E. coli suspension in M9-N D-glucose in M9-N volume/ [PC]/ n_pro/volume/ [Glu]/ OD₆₀₀ mL μM μmol mL mM 20 36.0 4.17 0.150 1.5 ~10 alkyne(1c) stock in EtOH EDA stock in EtOH volume/ n_1/ volume/ n_2/ stock/MμL mmol stock/M μL mmol 1.0 200 0.20 2.0 400 0.80 purification eluentproduct Ethyl acetate m[Pdt]/ n[Pdt]/ in hexanes mg mmol yield TTN (0%to 10% gradient) 45.2 0.146 73% 980

¹H NMR (400 MHz, CDCl₃) δ 7.28 (d, J=8.9 Hz, 2H), 7.23 (d, J=8.6 Hz,2H), 4.14 (qd, J=7.1, 2.4 Hz, 2H), 4.08 (qd, J=7.1, 2.2 Hz, 2H), 3.24(d, J=0.6 Hz, 1H), 3.10 (d, J=3.0 Hz, 1H), 3.08 (dd, J=3.0, 0.5 Hz, 1H),1.27 (t, J=7.1 Hz, 3H), 1.16 (t, J=7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃)δ 170.06, 167.75, 133.48, 131.27, 129.93, 128.71, 61.10, 61.06, 44.77,42.65, 27.47, 20.61, 14.36, 14.21. HRMS (FAB) m/z: 309.0902 (M+H⁺);calc. for C₁₆H₁₈Cl₄: 309.0894. [α]²³ _(D)=−141.5±2.6° (c 0.1, ethylacetate).

Diethyl (2R,4R)-1-(4-bromophenyl)bicyclo[1.1.0]butane-2,4-dicarboxylate(2d)

E. coli suspension in M9-N D-glucose in M9-N volume/ [PC]/ n_pro/volume/ [Glu]/ OD₆₀₀ mL μM μmol mL mM 20 36.0 4.17 0.150 1.5 ~10 alkyne(1d) stock in EtOH EDA stock in EtOH volume/ n_1/ volume/ n_2/ stock/MμL mmol stock/M μL mmol 1.0 150 0.15 2.0 300 0.60 purification eluentproduct Ethyl acetate m[Pdt]/ n[Pdt]/ in hexanes mg mmol yield TTN (0%to 10% gradient) 42.5 0.120 80% 800

¹H NMR (400 MHz, CDCl₃) δ 7.43 (d, J=8.7 Hz, 2H), 7.17 (d, J=8.7 Hz,2H), 4.14 (qd, J=7.1, 2.4 Hz, 2H), 4.08 (qd, J=7.1, 1.8 Hz, 2H), 3.24(d, J=0.6 Hz, 1H), 3.10 (d, J=3.1 Hz, 1H), 3.09 (dd, J=3.1, 0.5 Hz, 1H),1.27 (t, J=7.1 Hz, 3H), 1.16 (t, J=7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃)δ 170.02, 167.73, 131.84, 131.64, 130.22, 121.56, 61.11, 61.07, 44.74,42.62, 27.53, 20.71, 14.36, 14.21. HRMS (FAB) m/z: 353.0399 (M+H⁺);calc. for C₁₆H₁₈BrO₄: 353.0388 (⁷⁹Br). [α]²³ _(D)=−128.4±0.8° (c 0.1,ethyl acetate).

Diethyl (2R,4R)-1-(p-tolyl)bicyclo[1.1.0]butane-2,4-dicarboxylate (2e)

E. coli suspension in M9-N D-glucose in M9-N volume/ n_pro/ volume/[Glu]/ OD₆₀₀ mL [PC]/μM μmol mL mM 18 20.0 2.92 0.0584 1.0 ~13 alkyne(1e) stock in EtOH EDA stock in EtOH volume/ n_1/ volume/ n_2/ stock/MμL mmol stock/M μL mmol 1.0 160 0.16 2.0 160 0.32 purification eluentproduct Ethyl acetate m[Pdt]/ n[Pdt]/ in hexanes mg mmol yield TTN (0%to 8% gradient) 29.6 0.103 64% 1760

¹H NMR (400 MHz, CDCl₃) δ 7.19 (d, J=8.2 Hz, 2H), 7.11 (d, J=7.9 Hz,2H), 4.18-4.03 (m, 4H), 3.23 (d, J=0.6 Hz, 1H), 3.08 (dd, J=3.0, 0.6 Hz,1H), 3.06 (d, J=3.0 Hz, 1H), 2.32 (s, 3H), 1.26 (t, J=7.1 Hz, 3H), 1.15(t, J=7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 170.48, 168.01, 137.37,129.42, 129.22, 128.41, 60.94, 60.87, 44.62, 42.50, 28.09, 21.34, 20.07,14.37, 14.20. HRMS (FAB) m/z: 289.1450 (M+H⁺); calc. for C₁₇H₂₁O₄:289.1440. [α]²³ _(D)=−143.6±1.0° (c 0.1, ethyl acetate).

Diethyl (2R,4R)-1-(4-ethylphenyl)bicyclo[1.1.0]butane-2,4-dicarboxylate(2f)

E. coli suspension in M9-N D-glucose in M9-N volume/ n_pro/ volume/[Glu]/ OD₆₀₀ mL [PC]/μM μmol mL mM 18 32.0 2.92 0.0934 1.5 ~11 alkyne(1f) stock in EtOH EDA stock in EtOH volume/ n_1/ volume/ n_2/ stock/MμL mmol stock/M μL mmol 1.0 100 0.10 2.0 100 0.20 purification eluentproduct Ethyl acetate m[Pdt]/ n[Pdt]/ in hexanes mg mmol yield TTN (0%to 8% gradient) 16.8 0.0556 56% 600

¹H NMR (400 MHz, CDCl₃) δ 7.22 (d, J=8.3 Hz, 2H), 7.14 (d, J=8.4 Hz,2H), 4.20-4.01 (m, 4H), 3.23 (d, J=0.6 Hz, 1H), 3.09 (dd, J=3.0, 0.6 Hz,1H), 3.07 (d, J=3.0 Hz, 1H), 2.62 (q, J=7.6 Hz, 2H), 1.26 (t, J=7.1 Hz,3H), 1.21 (t, J=7.6 Hz, 3H), 1.14 (t, J=7.1 Hz, 3H). ¹³C NMR (101 MHz,CDCl₃) δ 170.49, 168.02, 143.73, 129.69, 128.46, 128.04, 60.93, 60.87,44.57, 42.50, 28.72, 28.13, 20.14, 15.67, 14.38, 14.19. HRMS (FAB) m/z:303.1588 (M+H⁺); calc. for C₁₈H₂₃O₄: 303.1596. [α]²³ _(D)=−133.7±1.8° (c0.1, ethyl acetate).

Diethyl(2R,4R)-1-(3-methoxyphenyl)bicyclo[1.1.0]butane-2,4-dicarboxylate (2g)

E. coli suspension in M9-N D-glucose in M9-N volume/ n_pro/ volume/[Glu]/ OD₆₀₀ mL [PC]/μM μmol mL mM 18 20.0 2.92 0.0584 1.0 ~13 alkyne(1g) stock in EtOH EDA stock in EtOH volume/ n_1/ volume/ stock/M μLmmol stock/M μL n_2/mmol 1.0 160 0.16 2.0 160 0.32 purification eluentproduct Ethyl acetate m[Pdt]/ n[Pdt]/ in hexanes mg mmol yield TTN (0%to 12% gradient) 27.2 0.0894 56% 1530

¹H NMR (400 MHz, CDCl₃) δ 7.24-7.19 (m, 1H), 6.88 (ddd, J=7.6, 1.6, 1.0Hz, 1H), 6.85-6.82 (m, 1H), 6.80 (ddd, J=8.2, 2.6, 1.0 Hz, 1H),4.20-4.01 (m, 4H), 3.78 (s, 3H), 3.25 (d, J=0.6 Hz, 1H), 3.13 (dd,J=3.0, 0.6 Hz, 1H), 3.09 (d, J=3.0 Hz, 1H), 1.26 (t, J=7.1 Hz, 3H), 1.15(t, J=7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 170.31, 167.90, 159.67,134.22, 129.54, 120.75, 114.01, 113.20, 61.01, 60.95, 55.35, 44.69,42.58, 28.18, 20.69, 14.37, 14.20. HRMS (FAB) m/z: 305.1383 (M+H⁺);calc. for C₁₇H₂₁O₅: 305.1389. [α]²³ _(D)=122.6±1.5° (c 0.1, ethylacetate).

Diethyl(2R,4R)-1-(3,5-dimethoxyphenyl)bicyclo[1.1.0]butane-2,4-dicarboxylate(2h)

E. coli suspension in M9-N D-glucose in M9-N volume/ n_pro/ volume/OD₆₀₀ mL [PC]/μM μmol mL [Glu]/mM 16 45.0 2.54 0.114 3.0 ~16 alkyne (1h)stock in EtOH EDA stock in EtOH n_1/ volume/ n_2/ stock/M volume/μL mmolstock/M μL mmol 1.0 150 0.15 2.0 225 0.45 purification eluent productEthyl acetate m[Pdt]/ n[Pdt]/ in hexanes mg mmol yield TTN (0% to 15%gradient) 19.8 0.0592 40% 520

¹H NMR (400 MHz, CDCl₃) δ 6.44 (d, J=2.2 Hz, 2H), 6.36 (t, J=2.3 Hz,1H), 4.15 (qd, J=7.1, 2.5 Hz, 2H), 4.09 (qd, J=7.1, 3.9 Hz, 2H),3.76 (s,6H), 3.24 (s, 1H), 3.12 (d, J=3.0 Hz, 1H), 3.09 (d, J=3.0 Hz, 1H), 1.26(t, J=7.1 Hz, 3H), 1.17 (t, J=7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ170.14, 167.76, 160.71, 134.80, 106.29, 99.71, 60.88, 60.84, 55.33,44.56, 42.45, 28.23, 20.67, 14.23, 14.11. HRMS (FAB) m/z: 335.1480(M+H^(f)); calc. for C₁₈H₂₃O₆: 335.1495. [α]²³ _(D)=144.6±3.5° (c 0.1,ethyl acetate).

Diethyl(2R,4R)-1-(3-hydroxyphenyl)bicyclo[1.1.0]butane-2,4-dicarboxylate (2i)

E. coli suspension in M9-N D-glucose in M9-N volume/ n_pro/ volume/[Glu]/ OD₆₀₀ mL [PC]/μM μmol mL mM 16 45.0 2.14 0.0963 3.0 ~16 alkyne(1i) stock in EtOH EDA stock in EtOH volume/ n_1/ volume/ n_2/ stock/MμL mmol stock/M μL mmol 1.0 120 0.12 2.0 180 0.36 purification eluentproduct Ethyl acetate n[Pdt]/ in hexanes m[Pdt]/mg mmol yield TTN (0% to20% gradient) 10.4 0.0358 30% 370

¹H NMR (400 MHz, CDCl₃) δ 7.15 (td, J=8.1, 1.7 Hz, 1H), 6.87-6.83 (m,1H), 6.78-6.74 (m, 1H), 6.73-6.68 (m, 1H), 5.06 (s, 1H), 4.21-4.02 (m,4H), 3.23 (d, J=0.6 Hz, 1H), 3.11 (dd, J=3.1, 0.6 Hz, 1H), 3.09 (d,J=3.0 Hz, 1H), 1.27 (t, J=7.1 Hz, 3H), 1.15 (t, J=7.1 Hz, 3H). ¹³C NMR(101 MHz, CDCl₃) δ 170.22, 167.87, 155.59, 134.27, 129.62, 120.76,115.15, 114.58, 60.94, 60.92, 44.54, 42.43, 27.86, 20.58, 14.23, 14.04.HRMS (FAB) m/z: 291.1237 (M+H⁺); calc. for C₁₆H₁₉O₅: 291.1232.

Diethyl (2R,4R)-1-(thiophen-2-yl)bicyclo[1.1.0]butane-2,4-dicarboxylate(2j)

E. coli suspension in M9-N D-glucose in M9-N n_pro/ volume/ [Glu]/ OD₆₀₀volume/mL [PC]/μM μmol mL mM 20 54.0 4.30 0.232 3.2 ~14 alkyne (1j)stock in EtOH EDA stock in EtOH volume/ n_1/ volume/ n_2/ stock/M μLmmol stock/M μL mmol 1.0 100 0.10 2.0 200 0.40 purification eluentproduct Ethyl acetate n[Pdt]/ in hexanes m[Pdt]/mg mmol yield TTN (0% to8% gradient) 11.4 0.0407 41% 160

¹H NMR (400 MHz, CDCl₃) δ 7.20 (dd, J=5.2, 1.2 Hz, 1H), 7.05 (dd, J=3.5,1.3 Hz, 1H), 6.94 (dd, J=5.2, 3.5 Hz, 1H), 4.19-4.10 (m, 4H), 3.27 (s,1H), 3.10 (d, J=3.1 Hz, 1H), 3.06 (dd, J=3.1, 0.6 Hz, 1H), 1.28 (t,J=7.1 Hz, 3H), 1.20 (t, J=7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ169.92, 167.46, 135.29, 128.55, 126.97, 125.42, 61.14, 61.12, 45.87,44.10, 23.88, 21.23, 14.37, 14.22. HRMS (FAB) m/z: 281.0839 (M+H⁺);calc. for C₁₄H₁₇O₄S: 281.0848.

Diethyl(2R,4R)-1-(4-methoxyphenyl)bicyclo[1.1.0]butane-2,4-dicarboxylate (2k)

E. coli suspension in M9-N D-glucose in M9-N volume/ n_pro/ volume/OD₆₀₀ mL [PC]/μM μmol mL [Glu]/mM 15 24.0 2.43 0.0583 1.0 ~10 alkyne(1k) stock in EtOH EDA stock in EtOH volume/ volume/ stock/M μL n_1/mmolstock/M μL n_2/mmol 1.0 160 0.16 2.0 160 0.32 purification eluentproduct Ethyl acetate m[Pdt]/ n[Pdt]/ in hexanes mg mmol yield TTN (0%to 8% gradient) 32.4 0.107 67% 1830 E. coli suspension in M9-N D-glucosein M9-N volume/ volume/ OD₆₀₀ mL [PC]/μM n_pro/μmol mL [Glu]/mM 16 6002.96 1.77 30 ~12 alkyne (1k) stock in EtOH EDA stock in EtOH volume/volume/ stock/M mL n_1/mmol stock/mM mL n_2/mmol 2.0 2.40 4.80 2.0 7.2014.40 purification eluent product Ethyl acetate n[Pdt]/ in hexanesm[Pdt]/mg mmol yield TTN (0% to 12% gradient) 680.4 2.34 47% 1260 Note:in 4.8 mmol scale reaction, the EDA stock solution was added in threeportions (2.4 mL each portion) every 1 hour.

¹H NMR (400 MHz, CDCl₃) δ 7.25 (d, J=8.7 Hz, 2H), 6.84 (d, J=8.7 Hz,2H), 4.20-3.99 (m, 4H), 3.79 (s, 3H), 3.21 (s, 1H), 3.04 (d, J=2.9 Hz,1H), 3.01 (d, J=2.9 Hz, 1H), 1.27 (t, J=7.1 Hz, 3H), 1.14 (t, J=7.1 Hz,3H). ¹³C NMR (101 MHz, CDCl₃) δ 170.53, 168.01, 159.21, 130.03, 124.27,113.99, 60.93, 60.85, 55.43, 44.68, 42.63, 27.90, 19.35, 14.38, 14.22.HRMS (FAB) m/z: 305.1375 (M+H⁺); calc. for C₁₇H₂₁O₅: 305.1389. [α]²³_(D)=138.2±1.6° (c 0.1, ethyl acetate).

Diethyl(2R,4R)-1-(4-ethynylxyphenyl)bicyclo[1.1.0]butane-2,4-dicarboxylate (2l)

The title compound was synthesized as summarized below. In the 2.8 mmolscale-reaction, EDA stock solution was added in three portions (1.4 mLeach portion) every 1 hour.

E. coli suspension in M9-N D-glucose in M9-N volume/ volume/ OD₆₀₀ mL[PC]/μM n_pro/μmol mL [Glu]/mM 16 36.0 3.44 0.124 2.0 ~13 alkyne (1l)stock in EtOH EDA stock in EtOH volume/ volume/ stock/M μL n_1/mmolstock/M μL n_2/mmol 1.0 150 0.15 2.0 225 0.45 purification eluentproduct Ethyl acetate n[Pdt]/ in hexanes m[Pdt]/mg mmol yield TTN (0% to8% gradient) 28.7 0.0962 64% 780 E. coli suspension in M9-N D-glucose inM9-N volume/ volume/ OD₆₀₀ mL [PC]/μM n_pro/μmol mL [Glu]/mM 18 600 3.031.82 30 ~12 alkyne (1l) stock in EtOH EDA stock in EtOH volume/ volume/stock/M mL n_1/mmol stock/M mL n_2/mmol 2.0 1.40 2.80 2.0 4.20 8.40purification eluent product Ethyl acetate n[Pdt]/ in hexanes m[Pdt]/mgmmol yield TTN (0% to 10% gradient) 393.7 1.32 47% 730 E. colisuspension in M9-N D-glucose in M9-N volume/ volume/ OD₆₀₀ mL [PC]/μMn_pro/μmol mL [Glu]/mM 18 600 3.03 1.82 30 ~12 alkyne (1l) stock in EtOHEDA stock in EtOH volume/ n_1/ volume/ stock/M mL mmol stock/M mLn_2/mmol 2.0 1.40 2.80 2.0 4.20 8.40 purification eluent product Ethylacetate n[Pdt]/ in hexanes m[Pdt]/mg mmol yield TTN (0% to 10% gradient)393.7 1.32 47% 730

¹H NMR (400 MHz, CDCl₃) δ 7.45 (d, J=8.5 Hz, 2H), 7.26 (d, J=8.6 Hz,2H), 4.17 (qd, J=7.2, 2.9 Hz, 2H), 4.11 (qd, J=7.1, 0.6 Hz, 2H), 3.29(d, J=0.6 Hz, 1H), 3.18 (dd, J=3.1, 0.6 Hz, 1H), 3.15 (d, J=3.1 Hz, 1H),3.11 (s, 1H), 1.29 (t, J=7.1 Hz, 3H), 1.17 (t, J=7.1 Hz, 3H). ¹³C NMR(101 MHz, CDCl₃) δ 170.04, 167.73, 133.79, 132.24, 128.28, 121.22,83.47, 77.86, 61.12, 61.07, 44.73, 42.67, 27.88, 21.40, 14.36, 14.19.HRMS (FAB) m/z: 299.1282 (M+H⁺); calc. for C₁₄H₁₉O₄: 299.1283. [α]²³_(D)=152.6±3.1° (c 0.1, ethyl acetate).

Diethyl(2R,4R)-1-(4-(hydroxymethyl)phenyl)bicyclo[1.1.0]butane-2,4-dicarboxylate(2m)

The title compound was prepared as summarized below. In the 1.0mmol-scale reaction, EDA stock solution was added in four portions (0.5mL each portion) every 1 hour.

E. coli suspension in M9-N D-glucose in M9-N volume/ volume/ OD₆₀₀ mL[PC]/μM n_pro/μmol mL [Glu]/mM 20 75.0 3.72 0.279 4.0 ~13 alkyne (1m)stock in EtOH EDA stock in EtOH volume/ volume/ stock/M μL n_1/mmolstock/M μL n_2/mmol 1.0 360 0.36 2.0 540 1.08 purification eluentproduct Ethyl acetate n[Pdt]/ in hexanes m[Pdt]/mg mmol yield TTN (0% to25% gradient) 52.8 0.174 48% 630 E. coli suspension in M9-N D-glucose inM9-N volume/ n_pro/ volume/ OD₆₀₀ mL [PC]/μM μmol mL [Glu]/mM 16 1602.68 0.428 10 ~15 alkyne (1m) stock in EtOH EDA stock in EtOH volume/volume/ stock/M mL n_1/mmol stock/M mL n_2/mmol 1.0 1.00 1.00 2.0 2.004.00 purification eluent product Ethyl acetate n[Pdt]/ in hexanesm[Pdt]/mg mmol yield TTN (0% to 25% gradient) 152.1 0.500 50% 580

¹H NMR (400 MHz, CDCl₃) δ 7.30 (d, J=2.5 Hz, 4H), 4.68 (d, J=5.0 Hz,2H), 4.14 (qd, J=7.1, 2.3 Hz, 2H), 4.08 (qd, J=7.1, 0.9 Hz, 2H), 3.26(d, J=0.5 Hz, 1H), 3.13 (dd, J=3.1, 0.6 Hz, 1H), 3.10 (d, J=3.0 Hz, 1H),1.62 (t, J=5.7 Hz, 1H), 1.27 (t, J=7.1 Hz, 3H), 1.16 (t, J=7.1 Hz, 3H).¹³C NMR (101 MHz, CDCl₃) δ 170.30, 167.93, 140.24, 132.11, 128.68,127.15, 65.20, 61.02, 60.96, 44.72, 42.54, 27.96, 20.58, 14.37, 14.22.HRMS (FAB) m/z: 303.1230 ((M+H⁺)−H₂); calc. for C₁₇H₁₉O₅: 303.1232.[α]²³ _(D)=−130.8±1.2° (c 0.1, ethyl acetate).

The enantioselectivities of bicyclobutane products are difficult todetermine, as there is no method in the literature to prepare theracemic standards. Determination of the enantiopurities of thebicyclobutane products by chiral HPLC was attempted. Purifiedbicyclobutanes 2a-2c, 2e, 2k and 2l (with >98% purity by ¹H NMR) weretested under various analytical chiral HPLC separation conditions usingwith Chiralpak IA, IC, AS-H, and OJ-H columns. In all cases, only onemajor peak was detected for each product (with >98% peak area at 254nm). In order to determine whether any of the minor peaks (with 0.5-1%peak area at 254 nm) detected corresponds to an enantiomer of thedesired product, those fractions were collected and concentrated forfurther UV testing (210-280 nm) and GC-MS testing; none turned out to bethe enantiomers of the bicyclobutanes. Though there is some possibilitythat the enantiomers might not be resolved under any of the chiral HPLCcondition tested, it is very likely that the bicyclobutanes have highenantiopurity.

Example 12 Engineering of Biocatalysts for Cyclopropene Formation

The next investigation focused on whether the enzyme could stop at thecyclopropene product if less reactive aliphatic alkynes are used. Tothis end, enzyme variants from the P411-S1 lineage were examined forcyclopropene formation, using phenylbutyne (5a) and EDA as startingreagents. It was encouraging to see that P4 catalyzed the desiredtransformation with 260 TTN and 95.5:4.5 er. Further evolution wasperformed on P4 to improve its catalytic efficiency. Position 87 wastargeted first, known for its importance to substrate recognition inP450-catalyzed oxidations (⁴⁶). A87F (290 TTN, 3.0:97.0 er) and A87W(240 TTN, 97.1:2.9 er) were found to exert the oppositeenantio-preference, suggesting that residue 87 also controls substrateorientation for non-native carbene chemistry. Single- anddouble-site-saturation mutagenesis conducted sequentially on P4 A87F andP4 A87W improved both reactivity and selectivity (FIG. 12A, FIG. 13 andFIG. 14). The final K10 and C6 variants performed with >10-fold higheractivity compared to the initial P4 variant and with excellentstereocontrol (99.55:0.45 er and 99.95:0.05 er, respectively).

Data from FIG. 13 and FIG. 14 are summarized in Table 7 and Table 8, andFIG. 15 and FIG. 16 depict the site targeted for mutation. Analyticalreactions were set up in quadruplicate according to the procedure inbiotransformations described above and using whole-cell catalyst andanalyzed by GC using the standard calibration curve of 6a. The TTNs wereobtained using OD₆₀₀=60 for P4 and K1 variants, OD₆₀₀=30 for K3, K5 andK6 variants, and OD₆₀₀=15 for K8 and K10 variants. TTNs reported are theaverage of biological duplicates. The TTNs were obtained using OD₆₀₀=60for P4 and C1 variants, OD₆₀₀=30 for C2, C3 and C4 variants, andOD₆₀₀=15 for C6 variant. TTNs reported are the average of fourexperiments. TTNs reported are the average of biological duplicates.Each biological set contains four experiments. The errors in all tablesare standard deviations.

TABLE 8 P411-P4 variants for the formation of cyclopropene (R)-6aP411-P4 variant TTN er P4  260 ± 30  95.5:4.5 P4 A87W (C1)  240 ± 40 97.1:2.9 K1 L437G (C2) 1400 ± 100 99.35:0.65 C2 V328Y (C3) 1600 ± 12099.80:0.20 C3 T327V (C4) 1700 ± 100 99.90:0.10 C4 F261M T436H (C6) 2500± 140 99.95:0.05

TABLE 9 Mutations in related P411-P4 variants Name Description Mutationsrelative to P450-WT P4 P411_(BM3)-CIS P4 variant (³⁴) V78A, F87A, P142S,T175I, A184V, S226R, H236Q, E252G, I263F, T268G, A290V, A328V, L353V,I366V, C400S, T438S, E442K K1 P411_(BM3)-CIS K1 variant V78A, P142S,T175I, A184V, S226R, H236Q, E252G, I263F, T268G, A290V, A328V, L353V,I366V, C400S, T438S, E442K K3 P411_(BM3)-CIS K3 variant V78A, P142S,T175I, A184V, S226R, H236Q, E252G, F261G, I263F, T268G, A290V, T327P,A328V, L353V, I366V, C400S, T438S, E442K K5 P411_(BM3)-CIS K5 variantS72W, V78A, P142S, T175I, A184V, S226R, H236Q, E252G, F261G, I263F,T268G, A290V, T327P, A328V, L353V, I366V, C400S, L437F, T438S, E442K K6P411_(BM3)-CIS K6 variant S72W, V78A, P142S, T175I, A184V, S226R, H236Q,E252G, F261G, I263F, T268G, T269L, A290V, T327P, A328V, L353V, I366V,C400S, L437F, T438S, E442K K8 P411_(BM3)-CIS K8 variant S72W, V78S,P142S, T175I, A184V, S226R, H236Q, E252G, F261G, I263F, T268G, T269L,A290V, T327P, A328V, A330V, L353V, I366V, C400S, L437F, T438S, E442K K10P411_(BM3)-CIS K10 variant S72W, V78S, P142S, T175I, A184V, L188C,S226R, H236Q, E252G, F261G, I263F, T268G, T269L, A290V, T327P, A328V,A330V, L353V, I366V, C400S, T436M, L437F, T438S, E442K

TABLE 10 Mutations in related P411-P4 variants Name DescriptionMutations relative to P450-WT P4 P411_(BM3)-CIS P4 variant ⁽³⁴) V78A,F87A, P142S, T175I, A184V, S226R, H236Q, E252G, I263F, T268G, A290V,A328V, L353V, I366V, C400S, T438S, E442K C1 P411_(BM3)-CIS C1 variantV78A, F87W, P142S, T175I, A184V, S226R, H236Q, E252G, I263F, T268G,A290V, A328V, L353V, I366V, C400S, T438S, E442K C2 P411_(BM3)-CIS C2variant V78A, F87W, P142S, T175I, A184V, S226R, H236Q, E252G, I263F,T268G, A290V, A328V, L353V, I366V, C400S, L437G, T438S, E442K C3P411_(BM3)-CIS C3 variant V78A, F87W, P142S, T175I, A184V, S226R, H236Q,E252G, I263F, T268G, A290V, A328Y, L353V, I366V, C400S, L437G, T438S,E442K C4 P411_(BM3)-CIS C4 variant V78A, F87W, P142S, T175I, A184V,S226R, H236Q, E252G, I263F, T268G, A290V, T327V, A328Y, L353V, I366V,C400S, L437G, T438S, E442K C6 P411_(BM3)-CIS C6 variant V78A, F87W,P142S, T175I, A184V, S226R, H236Q, E252G, F261M, I263F, T268G, A290V,T327V, A328Y, L353V, I366V, C400S, T436H, L437G, T438S, E442K

Example 13 Preparation of Alkyne Substrates

Commercially available substrates were used as received: all aromaticalkynes (1a-1m) (Sigma-Aldrich, Alfa-Aesar and Ark Pharm), aliphaticalkynes (5a-5c, 5g, 5j, 5k, 5m, and 5n) (Sigma-Aldrich, Alfa-Aesar andArk Pharm), ethyl 2-diazoacetate (Sigma-Aldrich).

Oct-7-yn-1-yl cyclobutanecarboxylate (5d). In a 100 mL round-bottomflask, oct-7-yn-1-ol (0.631 g, 5.0 mmol), triethylamine (0.9 mL, 1.3equiv.) and N,N-dimethylpyridin-4-amine (DMAP, 61 mg, 10 mol %) in dryDCM (20 mL) was cooled to 0° C. A solution of cyclobutanecarbonylchloride (0.711 g, 6.0 mmol) in dry DCM (5 mL) was added dropwise slowlyover 5 min. The reaction was allowed to warm to room temperature andstirred for 8 hours. The reaction mixture was diluted with DCM (20 mL)and washed with water (20 mL) and brine (20 mL), dried over MgSO₄, andthen concentrated under reduced pressure. The crude product was purifiedby silica column chromatography with hexane/ethyl acetate (20:1) toafford 5d (1.02 g, 98%). ¹H NMR (400 MHz, CDCl₃) δ 4.06 (t, J=6.7 Hz,2H), 3.12 (pd, J=8.5, 1.0 Hz, 1H), 2.34-2.13 (m, 6H), 2.04-1.83 (m, 3H),1.69-1.59 (m, 2H), 1.57-1.48 (m, 2H), 1.47-1.32 (m, 4H). ¹³C NMR (101MHz, CDCl₃) δ 175.62, 84.51, 68.25, 64.28, 38.17, 28.54, 28.34, 28.32,25.45, 25.28, 18.43, 18.33. HRMS (FAB) m/z: 209.1548 (M+H⁺); calc. forC₁₃H₂₁O₂: 209.1542.

2-(Hept-6-yn-1-yl)-1,3-dioxolane (5e). In a 250 mL round-bottom flask,DMSO (1.95 g, 25.0 mmol) was dissolved in dry DCM (40 mL) and thencooled to −78° C. under argon. A solution of oxalyl dichloride (2.6 mL,30.0 mmol) in dry DCM (10 mL) was added dropwise slowly over 10 min.After the mixture was stirred at −78° C. for 1 h, a solution ofoct-7-yn-1-ol (0.631 g, 5.0 mmol) in dry DCM (10 mL) was added dropwiseslowly over 10 min. The resulting mixture was maintained at −78° C. foranother 2 h before the dropwise addition of triethylamine (7.0 mL, 50.0mmol) in dry DCM (10 mL). The reaction was then allowed to warm to roomtemperature over 1 h and stirred for another 2 h. The reaction mixturewas diluted with DCM (30 mL) and washed with HCl (10%, 50 mL), NaHCO₃(50 mL, sat. aq.), and brine (50 mL), dried over MgSO₄, and thenconcentrated under reduced pressure (100 torr). The crude productmixture was used for the next step without further purification. Thecrude product was dissolved in benzene (50 mL) in a 100 mL round-bottomflask. Ethylene glycol (0.465 g, 7.5 mmol) and TsOH (130 mg, 0.075 mmol)were added. The solution was heated up to a refluxing temperature, 90°C., and maintained for 2 h. After removal of benzene under reducedpressure, the crude product was purified by silica column chromatographywith hexane/ether (20:1 to 15:1) to afford 5e (0.675 g, 80%). ¹H NMR(400 MHz, CDCl₃) δ 4.85 (t, J=4.8 Hz, 1H), 4.01-3.91 (m, 2H), 3.90-3.79(m, 2H), 2.19 (td, J=7.0, 2.6 Hz, 2H), 1.93 (t, J=2.7 Hz, 1H), 1.70-1.61(m, 2H), 1.59-1.51 (m, 2H), 1.49-1.39 (m, 4H). ¹³C NMR (101 MHz, CDCl₃)δ 104.66, 84.73, 68.32, 64.99, 33.89, 28.78, 28.53, 23.69, 18.45. HRMS(FAB) m/z: 167.1067 ((M+H⁺)−H₂); calc. for C₁₀H₁₅O₂: 167.1072.

5-Isopropoxypent-1-yne (5f). In a 100 mL round-bottom flask,pent-4-yn-1-ol (0.841 g, 10.0 mmol), triethylamine (1.8 mL, 1.3 equiv.)and DMAP (122 mg, 10 mol %) in dry DCM (50 mL) was cooled to 0° C.4-Methylbenzenesulfonyl chloride (TsCl, 2.10 g, 11.0 mmol) was addedportion-wise. The reaction was allowed to warm to room temperature andstirred for 6 hours. The reaction mixture was diluted with DCM (30 mL)and washed with water (20 mL) and brine (20 mL), dried over MgSO₄, andthen concentrated under reduced pressure. The crude product was purifiedby silica column chromatography with hexane: ethyl acetate (4:1) toafford pent-4-yn-1-yl 4-methylbenzenesulfonate (2.35 g, 99%).

Isopropanol (0.361 g, 6.0 mmol) and tetrabutylammonium bromide (161 mg,0.5 mmol) were dissolved in anhydrous DMF (50 mL) and then cooled to 0°C. NaH (60%, 240 mg, 6.0 mmol) was added portion-wise to the solution.The resulting mixture was stirred at 0° C. for 1 h before the dropwiseaddition of pent-4-yn-1-yl 4-methylbenzenesulfonate (1.19 g, 5.0 mmol)in anhydrous DMF (10 mL). The reaction was allowed to warm to roomtemperature and stirred for 4 hours. The reaction mixture was quenchedwith NH₄Cl (20 mL, sat. aq.) and the product was extracted with Et₂O (30mL×3). The combined organic layer was washed with water (20 mL), brine(20 mL), dried over MgSO₄, and then concentrated under reduced pressure(200 ton). The crude product was purified by silica columnchromatography with pentane/ether (10:1) to afford 5f (0.597 g, 95%).

4((Oct-7-yn-1-yloxy)methyl)tetrahydro-2H-pyran (5h). In a 500 mLround-bottom flask, oct-7-yn-1-ol (6.31 g, 50.0 mmol), triethylamine(9.1 mL, 1.3 equiv.) and DMAP (609 mg, 10 mol %) in dry DCM (200 mL) wascooled to 0° C. TsCl (10.50 g, 55.0 mmol) was added portion-wise. Thereaction was allowed to warm to room temperature and stirred for 6hours. The reaction mixture was diluted with DCM (100 mL) and washedwith water (200 mL) and brine (200 mL), dried over MgSO₄, and thenconcentrated under reduced pressure. The crude product was purified bysilica column chromatography with hexane: ethyl acetate (10:1) to affordoct-7-yn-1-yl 4-methylbenzenesulfonate (13.29 g, 95%).

In a 100 mL round-bottom flask, (tetrahydro-2H-pyran-4-yl)methanol(0.697 g, 6.0 mmol) and tetrabutylammonium bromide (161 mg, 0.5 mmol)were dissolved in anhydrous DMF (50 mL) and then cooled to 0° C. NaH(60%, 240 mg, 6.0 mmol) was added portion-wise to the solution. Theresulting mixture was stirred at 0° C. for 1 h before the dropwiseaddition of oct-7-yn-1-yl 4-methylbenzenesulfonate (1.40 g, 5.0 mmol) inanhydrous DMF (10 mL). Then reaction was allowed to warm to roomtemperature and stirred for 4 hours. The reaction mixture was quenchedwith NH₄Cl (20 mL, sat. aq.) and the product was extracted with Et₂O (50mL×3). The combined organic layer was washed with water (20 mL), brine(20 mL), dried over MgSO₄, and then concentrated under reduced pressure.The crude product was purified by silica column chromatography withhexane/ethyl acetate (10:1) to afford 5h (1.10 g, 98%). ¹H NMR (400 MHz,CDCl₃) δ 3.96 (ddt, J=11.5, 4.6, 1.1 Hz, 2H), 3.43-3.34 (m, 4H), 3.24(d, J=6.6 Hz, 2H), 2.18 (td, J=7.0, 2.7 Hz, 2H), 1.93 (t, J=2.7 Hz, 1H),1.89-1.77 (m, 1H), 1.68-1.61 (m, 2H), 1.60-1.49 (m, 4H), 1.46-1.34 (m,4H), 1.33-1.25 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 84.80, 76.11, 71.24,68.28, 67.90, 35.60, 30.19, 29.70, 28.70, 28.56, 25.83, 18.49. HRMS(FAB) m/z: 225.1855 (M+H⁺); calc. for C₁₄H₂₅O₂: 225.1855.

((Oct-7-yn-1-yloxy)methyl)cyclopropane (5i). In a 100 mL round-bottomflask, cyclopropylmethanol (0.433 g, 6.0 mmol) and tetrabutylammoniumbromide (161 mg, 0.5 mmol) were dissolved in anhydrous DMF (50 mL) andthen cooled to 0° C. NaH (60%, 240 mg, 6.0 mmol) was added portion-wiseto the solution. The resulting mixture was stirred at 0° C. for 1 hbefore the dropwise addition of oct-7-yn-1-yl 4-methylbenzenesulfonate(1.40 g, 5.0 mmol) in anhydrous DMF (10 mL). The reaction was allowed towarm to room temperature and stirred for 4 hours. The reaction mixturewas quenched with NH₄Cl (20 mL, sat. aq.) and the product was extractedwith Et₂O (50 mL×3). The combined organic layer was washed with water(20 mL), brine (20 mL), dried over MgSO₄, and then concentrated underreduced pressure. The crude product was purified by silica columnchromatography with hexane/ether (50:1) to afford 5i (840 mg, 93%). ¹HNMR (400 MHz, CDCl₃) δ 3.42 (t, J=6.7 Hz, 2H), 3.24 (d, J=6.9 Hz, 2H),2.18 (td, J=7.0, 2.6 Hz, 2H), 1.93 (t, J=2.6 Hz, 1H), 1.64-1.57 (m, 2H),1.56-1.49 (m, 2H), 1.47-1.30 (m, 4H), 1.12-0.97 (m, 1H), 0.58-0.45 (m,2H), 0.19 (dt, J=6.0, 4.5 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 84.82,75.73, 70.77, 68.26, 29.78, 28.72, 28.57, 25.85, 18.49, 10.82, 3.13.HRMS (FAB) m/z: 181.1590 (M+H⁺); calc. for C₁₂H₂₁O: 181.1592.

Trans-1-(methoxymethyl)-4-((oct-7-yn-1-yloxy)methyl)cyclohexane (51). Ina 100 mL round-bottom flask, (trans-cyclohexane-1,4-diyl)dimethanol(2.88 g, 20.0 mmol) and tetrabutylammonium bromide (32 mg, 0.1 mmol)were dissolved in anhydrous DMF (50 mL) and then cooled to 0° C. NaH(60%, 960 mg, 24.0 mmol) was added portion-wise to the solution. Theresulting mixture was stirred at 0° C. for 1 h before the dropwiseaddition of methyl iodide (MeI, 1.5 mL, 24.0 mmol) in anhydrous DMF (10mL). The reaction was allowed to warm to room temperature and stirredfor 4 hours. The reaction mixture was quenched with NH₄Cl (20 mL, sat.aq.) and the product was extracted with Et₂O (50 mL×4). The combinedorganic layer was washed with water (50 mL), brine (50 mL), dried overMgSO₄, and then concentrated under reduced pressure. The crude productwas purified by silica column chromatography with hexane/ethyl acetate(1:1) to afford (trans-4-(methoxymethyl)cyclohexyl)methanol (2.02 g,64%).

In a 100 mL round-bottom flask,(trans-4-(methoxymethyl)cyclohexyl)methanol (0.949 g, 6.0 mmol) andtetrabutylammonium bromide (161 mg, 0.5 mmol) were dissolved inanhydrous DMF (50 mL) and then cooled to 0° C. NaH (60%, 240 mg, 6.0mmol) was added portion-wise to the solution. The resulting mixture wasstirred at 0° C. for 1 h before the dropwise addition of oct-7-yn-1-yl4-methylbenzenesulfonate (1.40 g, 5.0 mmol) in anhydrous DMF (10 mL).Then reaction was allowed to warm to room temperature and stirred for 4hours. The reaction mixture was quenched with NH₄Cl (20 mL, sat. aq.)and the product was extracted with Et₂O (50 mL×3). The combined organiclayer was washed with water (20 mL), brine (20 mL), dried over MgSO₄,and then concentrated under reduced pressure. The crude product waspurified by silica column chromatography with hexane/ethyl acetate(10: 1) to afford 51 (743 mg, 56%). ¹H NMR (400 MHz, CDCl₃) δ 3.38 (t,J=6.6 Hz, 2H), 3.32 (s, 3H), 3.20 (d, J=6.5 Hz, 2H), 3.18 (d, J=6.4 Hz,2H), 2.18 (td, J=7.0, 2.7 Hz, 2H), 1.93 (t, J=2.7 Hz, 1H), 1.86-1.74 (m,4H), 1.58-1.49 (m, 6H), 1.46-1.30 (m, 4H), 1.01-0.86 (m, 4H). ¹³C NMR(101 MHz, CDCl₃) δ 84.84, 78.93, 76.90, 71.17, 68.26, 58.98, 38.37 (2carbons), 29.73, 29.65, 29.55, 28.73, 28.58, 25.85, 18.50. HRMS (FAB)m/z: 267.2325 (M+H⁺); calc. for C₁₇H₃₁O₂: 267.2324.

Example 14 Analytical-scale and preparative-scale enzymatic cyclopropeneformation

To evaluate the substrate range of the evolved P411 variants forcyclopropene construction, P411-C6 activity was examined usingstructurally diverse aliphatic alkynes. Enzymatic reactions with 12alkynes in preparative scale (up to 5.0 mmol scale) afforded the desiredcyclopropenes with TTNs ranging from hundreds to thousands and good toexcellent stereoselectivities (FIG. 3B and 3C). Alkynes with a linearcarbon chain (5b) or cyclic fragments (5g, 5h and 5j) all served as goodsubstrates. Different functional groups, including ether (5f, 5i and5l), ester (5d), acetal (5e), chloride (5k), and free hydroxyl (5m),were well-tolerated. Further optimization of reaction conditions withslow addition of EDA, for example, would likely improve the isolatedyields, as was demonstrated for cyclopropene 6h (66% yield, FIG. 3B; and94% yield, FIG. 3C).

All enzymatic reactions for cyclopropene formation in analytical scalewere conducted following the general procedure described below andanalyzed with gas chromatography (GC). All TTNs for the differentproducts were determined using the GC standard curve of thecorresponding racemic standard product made with Rh₂(OAc)₄ (see SectionVII). These TTNs were used to guide set-up of preparative-scalereactions.

General procedure for analytical-scale reactions: To a 2 mL vial wereadded degassed suspension of E. coli expressing P411-C6 or K10 variantin M9-N buffer (OD₆₀₀=15, 340 μL), alkyne (10 μL of 400 or 800 mM stocksolution in EtOH, 10 or 20 mM), EDA (10 μL of 400 or 800 mM stocksolution in EtOH, 10 or 20 mM, 1.0 equiv.), D-glucose (40 μL of 250 mMstock solution in M9-N buffer, 25 mM) under anaerobic conditions. Thevial was capped and shaken at 560 rpm at room temperature for 6 h. Afterthe reaction was completed, internal standard 1,3,5-trimethoxybenzene(20 μL of 20 mM stock solution in toluene) was added to the reactionvial followed by mixed solvent (cyclohexane/ethyl acetate=1:1, 1 mL).The mixture was transferred to a 1.5 mL microcentrifuge tube, and thenvortexed (15 seconds×3) and centrifuged (14,000 rpm, 5 min) tocompletely separate the organic and aqueous layers. 0.8 mL of organiclayer was taken for GC analysis. TTN was calculated based on measuredprotein concentration. Enantioselectivity was measured by chiral HPLC.Reactions for every substrate were set up in triplicate orquadruplicate.

GC standard curve: All data points represent the average of duplicateruns. The standard curves plot product concentration in mM (y-axis)against the ratio of product area to internal standard area on the GC(x-axis).

All enzymatic reactions for cyclopropene formation in preparative scalewere conducted following the general procedure described below and thecorresponding cyclopropene products were isolated. Detailed conditionsfor preparative-scale reactions of different substrates are indicatedseparately.

General procedure for preparative-scale reactions. To a 40 mL vial or250 mL flask were added degassed suspension of E. coli expressingP411-C6 or K10 variant (OD₆₀₀=10-60), alkyne (0.08-0.4 mmol, largerscales for 5a and 5h), EDA (1.0-4.0 equiv.), D-glucose (10-15 mM, 250 mMstock in M9-N), 1-5 vol % EtOH, M9-N buffer (pH 7.4) under anaerobicconditions. The vial or flask was capped and shaken (420 rpm for vialsand 220 rpm for flasks) at room temperature for 12 h.

After the reaction was completed, every 30 mL portion ofpreparative-scale reaction mixture was transferred to a 50 mL Falconcentrifuge tube. The reaction container was washed with water (2 mL×2)followed by mixed organic solvent (cyclohexane/ethyl acetate=1:1, 2mL×3). The washing solution was combined to reaction mixture incentrifuge tubes. An additional 12 mL of cyclohexane/ethyl acetatesolvent was added to every tube. After the tube (with ˜45 mL mixture intotal) was capped, it was vortex (1 min×3) and shaken vigorously, andcentrifuged (14,000 g, 5 min). The organic layer was separated and theaqueous layer was subjected to three more rounds of extraction. Theorganic layers were combined, dried over Na₂SO₄ and concentrated underreduced pressure. Purification by silica column chromatography withhexane/ethyl acetate as eluent afforded the desired cyclopropenes.Enantioselectivity was measured by chiral HPLC. TTNs were calculatedbased on measured protein concentration and isolated product yield.

Ethyl (S)-2-phenethylcycloprop-2-ene-1-carboxylate (S-6a)

Analysis data (10 mM 5a, P411-K10):

Pdt- [Pdt]/ [PC]/ Avg. Avg. entries Pdt Std Pdt/Std mM μM TTN TTN yield6a-(1) 4637.1 590.6 7.852 6.24 1.93 3242 6a-(2) 4639.5 592.1 7.836 6.231.93 3236 6a-(3) 4797.4 582.9 8.230 6.54 1.93 3399 6a-(4) 4674.5 588.07.950 6.32 1.93 3283 3290 63.3%Preparative-scale reaction:

E. coli suspension expressing P411-K10 in M9-N D-glucose in M9-N OD₆₀₀volume/mL [PC]/μM n_pro/μmol volume/mL [Glu]/mM 20 28.0 3.12 0.0874 1.5~13 alkyne (5a) stock in EtOH EDA stock in EtOH stock/ n_1/ n_2/ Mvolume/μL mmol stock/M volume/μL mmol 0.80 500 0.40 0.80 500 0.40purification eluent product ethyl acetate m[Pdt]/ n[Pdt]/ in hexanes mgmmol yield TTN (0% to 5% gradient) 37.8 0.175 44% 2000

¹H NMR (400 MHz, CDCl₃) δ 7.33-7.27 (m, 2H), 7.24-7.18 (m, 3H), 6.36(dt, J=1.4 Hz, 1H), 4.21-4.05 (m, 2H), 2.96-2.79 (m, 4H), 2.14 (d, J=1.5Hz, 1H), 1.25 (t, J=7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 176.61,140.81, 128.58, 128.45, 126.36, 115.01, 95.03, 60.39, 33.00, 26.90,20.00, 14.52. HRMS (FAB) m/z: 217.1221 (M+H⁺); calc. for C₁₄H₁₇O₂:217.1229. [α]²³ _(D)=−64.7±3.6° (c 0.1, cyclohexane). HPLC Chiralpak ICcolumn (n-hexane/isopropanol=95:5, 1.0 mL/min), tr=8.89 min (minor),9.20 min (major), 99.55:0.45 er.

Ethyl (R)-2-phenethylcycloprop-2-ene-1-carboxylate (R-6a)

Analysis data (10 mM 5a, P411-C6):

Pdt- [Pdt]/ [PC]/ Avg. Avg. entries Pdt Std Pdt/Std mM μM TTN TTN yield6a-(1) 4577.9 511.2 8.955 7.12 2.83 2518 6a-(2) 4659.1 515.1 9.045 7.192.83 2543 6a-(3) 4647.7 518.4 8.965 7.13 2.83 2521 6a-(4) 4608.7 518.68.887 7.07 2.83 2499 2520 71.3%Preparative-scale reaction:

E. coli suspension expressing P411-C6 in M9-N D-glucose in M9-N volume/n_pro/ volume/ [Glu]/ OD₆₀₀ mL [PC]/μM μmol mL mM 20 28.0 4.04 0.113 1.5~13 alkyne (5a) stock in EtOH EDA stock in EtOH stock/ volume/ n_1/ n_2/M μL mmol stock/M volume/μL mmol 0.80 500 0.40 0.80 500 0.40purification eluent product Ethyl acetate m[Pdt]/ n[Pdt]/ in hexanes mgmmol yield TTN (0% to 5% gradient) 46.8 0.216 54% 1910 E. colisuspension expressing P411-C6 in M9-N D-glucose in M9-N volume/ n_pro/volume/ [Glu]/ OD₆₀₀ mL [PC]/μM μmol mL mM 24 800 5.35 4.28 50 ~15alkyne (5a) stock in EtOH EDA stock in EtOH volume/ n_1/ volume/ n_2/stock/M mL mmol stock/M mL mmol 2.0 2.50 5.0 2.0 7.50 15.0 purificationeluent product Ethyl acetate m[Pdt]/ n[Pdt]/ in hexanes mg mmol yieldTTN (0% to 5% gradient) 824.1 3.81 76% 1780

In the 5.0-mmol scale reaction, EDA stock solution was added in threeportions (2.5 mL each portion) every 1 hour. [α]²³ _(D)=−62.2±2.5° (c0.1, cyclohexane). HPLC Chiralpak IC column (n-hexane/isopropanol=95:5,1.0 mL/min), tr=9.05 min (major), 9.42 min (minor), 99.95:0.05 er.

Ethyl (R)-2-octylcycloprop-2-ene-1-carboxylate (6b)

Analysis data (10 mM 5b, P411-C6):

Pdt- [Pdt]/ [PC]/ Avg. Avg. entries Pdt Std Pdt/Std mM μM TTN TTN yield6b-(1) 1195.2 523.7 2.282 1.49 1.82 815 6b-(2) 1128.3 528.7 2.134 1.391.82 763 6b-(3) 1023.1 521.4 1.962 1.28 1.82 701 760 13.9%Preparative-scale reaction:

E. coli suspension expressing P411-C6 in M9-N D-glucose in M9-N volume/n_pro/ volume/ [Glu]/ OD₆₀₀ mL [PC]/μM μmol mL mM 20 60.0 3.83 0.230 1.5~12 alkyne (5b) stock in EtOH EDA stock in EtOH volume/ n_1/ volume/stock/M μL mmol stock/M μL n_2/mmol 0.80 250 0.20 0.80 250 0.20purification eluent product Ethyl acetate m[Pdt]/ n[Pdt]/ in hexanes mgmmol yield TTN (0% to 5% gradient) 27.2 0.121 61% 530

The cell suspension (OD₆₀₀=20) was added in three portions (20 mL eachportion) every 2 hours.

¹H NMR (400 MHz, CDCl₃) δ 6.31 (dt, J=1.4 Hz, 1H), 4.12 (qd, J=7.1, 5.0Hz, 2H), 2.48 (td, J=7.3, 1.4 Hz, 2H), 2.12 (d, J=1.6 Hz, 1H), 1.61-1.52(m, 2H), 1.35-1.22 (m, 13H), 0.91-0.83 (m, 3H). ¹³C NMR (101 MHz, CDCl₃)δ 176.80, 115.77, 94.01, 60.28, 31.97, 29.39, 29.32, 29.27, 26.80,25.10, 22.79, 19.86, 14.52, 14.24. HRMS (FAB) m/z: 225.1862 (M+H⁺);calc. for C₁₄H₂₅O₂: 225.1855. [α]²³ _(D)=+32.6±1.8° (c 0.1, cyclohexane)[Note: (S)-5b (87% ee) was reported with [α]²³ _(D)=−30° (c 1.55, CHCl₃)(²²)]. HPLC Chiralpak IC column (n-hexane/isopropanol=95:5, 1.0 mL/min),tr=6.59 min (major), 7.10 min (minor), 98.0:2.0 er.

Ethyl (R)-2-(hex-5-yn-1-yl)cycloprop-2-ene-1-carboxylate (6c)

Analysis data (20 mM 5c, P411-C6):

Pdt- [Pdt]/ [PC]/ Avg. Avg. entries Pdt Std Pdt/Std mM μM TTN TTN yield6c-(1) 1084.2 344.6 3.146 2.39 1.67 1434 6c-(2) 1164.4 407.1 2.860 2.171.67 1304 6c-(3) 1079.9 385.7 2.800 2.13 1.67 1276 6c-(4) 985.5 350.12.815 2.14 1.67 1283 1324 11.1%Preparative-scale reaction:

E. coli suspension expressing P411-C6 in M9-N D-glucose in M9-N volume/n_pro/ volume/ [Glu]/ OD₆₀₀ mL [PC]/μM μmol mL mM 30 30.0 6.33 0.190 1.5~12 alkyne (5c) stock in EtOH EDA stock in EtOH volume/ n_1/ volume/n_2/ stock/M μL mmol stock/M μL mmol 0.80 500 0.40 0.80 500 0.40purification eluent product Ethyl acetate m[Pdt]/ n[Pdt]/ in hexanes mgmmol yield TTN (0% to 6% gradient) 46.2 0.240 60% 1260

¹H NMR (400 MHz, CDCl₃) δ 6.35 (dt, J=1.5 Hz, 1H), 4.20-4.04 (m, 2H),2.52 (td, J=7.2, 1.4 Hz, 2H), 2.21 (td, J=6.9, 2.6 Hz, 2H), 2.13 (d,J=1.6 Hz, 1H), 1.94 (t, J=2.6 Hz, 1H), 1.71 (dtd, J=8.7, 7.1, 5.4 Hz,2H), 1.60 (dtd, J=9.1, 6.9, 4.9 Hz, 2H), 1.24 (t, J=7.1 Hz, 3H). ¹³C NMR(101 MHz, CDCl₃) δ 176.63, 115.25, 94.60, 84.14, 68.69, 60.33, 27.89,25.84, 24.63, 19.85, 18.23, 14.51. HRMS (FAB) m/z: 193.1220 (M+H⁺);calc. for C₁₂H₁₇O₂: 193.1229. [α]²³ _(D)=+52.2±1.8° (c 0.1,cyclohexane). HPLC Chiralpak IC column (n-hexane/isopropanol=95:5, 1.0mL/min), tr=10.37 min (major), 11.55 min (minor), 98.0:2.0 er.

(R)-6-(3-(Ethoxycarbonyl)cycloprop-1-en-1-yl)hexylcyclobutanecarboxylate (6d)

Analysis data (10 mM 5d, P411-C6):

Pdt- [Pdt]/ Avg. Avg. entries Pdt Std Pdt/Std mM [PC]/μM TTN TTN yield6d-(1) 24642 4192 5.878 3.88 2.01 1930 6d-(2) 22575 4190 5.388 3.56 2.011769 6d-(3) 22572 4242 5.321 3.51 2.01 1747 6d-(4) 24260 4249 5.710 3.772.01 1875 1831 36.8%Preparative-scale reaction:

E. coli suspension expressing P411-C6 in M9-N D-glucose in M9-N volume/n_pro/ volume/ OD₆₀₀ mL [PC]/μM μmol mL [Glu]/mM 32 25.0 6.48 0.162 1.5~11 alkyne (5d) stock in EtOH EDA stock in EtOH volume/ n_1/ volume/stock/M μL mmol stock/M μL n_2/mmol 1.0 360 0.36 1.0 900 0.90purification eluent product Ethyl acetate m[Pdt]/ n[Pdt]/ in hexanes mgmmol yield TTN (0% to 12% gradient) 56.7 0.193 54% 1190

¹H NMR (400 MHz, CDCl₃) δ 6.33 (dt, J=1.5 Hz, 1H), 4.17-4.08 (m, 2H),4.05 (t, J=6.7 Hz, 2H), 3.12 (p, J=8.5 Hz, 1H), 2.49 (td, J=7.3, 1.1 Hz,2H), 2.33-2.13 (m, 4H), 2.12 (d, J=1.6 Hz, 1H), 2.05-1.82 (m, 2H),1.66-1.55 (m, 4H), 1.43-1.32 (m, 4H), 1.25 (t, J=7.1 Hz, 3H). ¹³C NMR(101 MHz, CDCl₃) δ 176.74, 175.74, 115.55, 94.26, 64.38, 60.33, 38.29,28.87, 28.68, 26.68, 25.74, 25.42, 25.03, 19.85, 18.56, 14.54. HRMS(FAB) m/z: 295.1910 (M+H⁺); calc. for C₁₇H₂₇O₄: 295.1909. [α]²³_(D)=+31.7±1.7° (c 0.1, cyclohexane). HPLC Chiralpak IC column(n-hexane/isopropanol=91:9, 1.0 mL/min), tr=17.06 min (major), 19.06 min(minor), 95.3:4.7 er.

Ethyl (R)-2-(5-(1,3-dioxolan-2-yl)pentyl)cycloprop-2-ene-1-carboxylate(6e)

Analysis data (10 mM 5e, P411-C6):

Pdt- [Pdt]/ Avg. Avg. entries Pdt Std Pdt/Std mM [PC]/μM TTN TTN yield6e-(1) 7788 4377 1.779 1.48 1.77 838 6e-(2) 7579 4283 1.770 1.47 1.77834 6e-(3) 7654 4377 1.749 1.46 1.77 824 6e-(4) 7263 4296 1.691 1.411.77 797 823 14.6%Preparative-scale reaction:

E. coli suspension expressing P411-C6 in M9-N D-glucose in M9-N n_pro/volume/ [Glu]/ OD₆₀₀ volume/mL [PC]/μM μmol mL mM 24 50.0 5.32 0.266 2.0~10 alkyne (5e) stock in EtOH EDA stock in EtOH n_1/ volume/ n_2/stock/M volume/μL mmol stock/M μL mmol 1.0 240 0.24 1.0 720 0.72purification eluent product Ethyl acetate m[Pdt]/ n[Pdt]/ in hexanes mgmmol yield TTN (0% to 15% gradient) 42.8 0.168 70% 630

The cell suspension (OD₆₀₀=24) was added in two portions (25 mL eachportion) every 2 hours.

¹H NMR (400 MHz, Acetone-d6) δ 6.61 (dt, J=1.5 Hz, 1H), 4.81 (t, J=4.8Hz, 1H), 4.09 (qd, J=7.1, 2.3 Hz, 2H), 3.98-3.88 (m, 2H), 3.87-3.76 (m,2H), 2.54 (tdd, J=7.1, 2.2, 1.4 Hz, 2H), 2.07 (d, J=1.6 Hz, 1H),1.67-1.57 (m, 4H), 1.52-1.41 (m, 4H), 1.23 (t, J=7.1 Hz, 3H). ¹³C NMR(101 MHz, Acetone-d6) δ 175.78, 115.81, 104.67, 94.74, 65.05, 59.93,34.29, 27.09, 25.03, 24.19, 19.69, 14.44 (one carbon peak may beoverlapping with the solvent peaks). HRMS (FAB) m/z: 255.1593 (M+H⁺);calc. for C₁₄H₂₃O₄: 255.1596. [α]²³ _(D)=+29.8±1.3° (c 0.1,cyclohexane). HPLC Chiralpak IC column (n-hexane/isopropanol=95:5, 1.0mL/min), tr=15.03 min (major), 16.92 min (minor), 96.8:3.2 er.

Ethyl (R)-2-(3-isopropoxypropyl)cycloprop-2-ene-1-carboxylate (6f)

Analysis data (10 mM 5f, P411-C6):

Pdt- [Pdt]/ Avg. Avg. entries Pdt Std Pdt/Std mM [PC]/μM TTN TTN yield6f-(1) 212.9 504.7 0.422 0.38 1.82 207 6f-(2) 289.9 514.0 0.564 0.501.82 276 6f-(3) 252.4 511.2 0.494 0.44 1.82 242 242 4.4%Preparative-scale reaction:

E. coli suspension D-glucose expressing P411-C6 in M9-N in M9-N n_pro/volume/ [Glu]/ OD₆₀₀ volume/mL [PC]/μM μmol mL mM 30 40.0 6.27 0.251 2.0~12 alkyne (5f) stock in EtOH EDA stock in EtOH n_1/ volume/ n_2/stock/mM volume/μL mmol stock/mM μL mmol 800 100 0.08 800 300 0.24product purification eluent m[Pdt]/ n[Pdt]/ Ethyl acetate in hexanes mgmmol yield TTN (0% to 8% gradient) 13.6 0.0641 80% 260

¹H NMR (400 MHz, CDCl₃) δ 6.34 (dt, J=1.4 Hz, 1H), 4.12 (qd, J=7.1, 4.2Hz, 2H), 3.53 (hept, J=6.2 Hz, 1H), 3.45 (td, J=6.3, 1.9 Hz, 2H), 2.58(td, J=7.3, 1.4 Hz, 2H), 2.13 (d, J=1.5 Hz, 1H), 1.88-1.78 (m, 2H), 1.24(t, J=7.1 Hz, 3H), 1.13 (d, J=6.1 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ176.69, 115.36, 94.47, 71.61, 66.92, 60.31, 27.43, 22.24, 22.02, 19.89,14.51. HRMS (FAB) m/z: 213.1485 (M+H⁺); calc. for C₁₂H₂₁O₃: 213.1491.HPLC Chiralpak IC column (n-hexane/isopropanol=95:5, 1.0 mL/min),tr=9.42 min (major), 10.92 min (minor), 97.3:2.7 er.

Ethyl (R)-2-(cyclohexylmethyl)cycloprop-2-ene-1-carboxylate (6g)

Analysis data (20 mM 5g, P411-C6):

Pdt- [Pdt]/ Avg. Avg. entries Pdt Std Pdt/Std mM [PC]/μM TTN TTN yield6g-(1) 1044.5 704.1 1.483 0.97 1.67 585 6g-(2) 903.7 654.8 1.380 0.911.67 544 6g-(3) 1102.7 701.6 1.572 1.03 1.67 620 6g-(4) 1105.0 726.71.521 1.00 1.67 600 587 4.9%Preparative-scale reaction:

E. coli suspension expressing P411-C6 in M9-N D-glucose in M9-N n_pro/volume/ [Glu]/ OD₆₀₀ volume/mL [PC]/μM μmol mL mM 20 60.0 3.83 0.230 1.5~12 alkyne (5g) stock in EtOH EDA stock in EtOH stock/M volume/μLn_1/mmol stock/M volume/μL n_2/mmol 0.80 250 0.20 0.80 500 0.40purification eluent product Ethyl acetate m[Pdt]/ n[Pdt]/ in hexanes mgmmol yield TTN (0% to 5% gradient) 23.1 0.111 55% 480

The cell suspension (OD₆₀₀=20) was added in three portions (20 mL eachportion) every 2 hours.

¹H NMR (400 MHz, CDCl₃) δ 6.33 (d, J=1.4 Hz, 1H), 4.12 (qd, J=7.1, 3.3Hz, 2H), 2.38 (d, J=7.2 Hz, 2H), 2.10 (d, J=1.6 Hz, 1H), 1.82-1.52 (m,6H), 1.27-1.07 (m, 6H), 0.98 (qt, J=13.6, 3.7 Hz, 2H). ¹³C NMR (101 MHz,CDCl₃) δ 176.76, 114.74, 94.58, 60.28, 36.22, 33.18, 33.17, 32.68,26.41, 26.27, 26.25, 19.93, 14.53. HRMS (FAB) m/z: 209.1537 (M+H⁺);calc. for C₁₃H₂₁O₂: 209.1542. [α]²³ _(D)=+53.4±1.5° (c 0.1,cyclohexane). HPLC Chiralpak IC column (n-hexane/isopropanol=95:5, 1.0mL/min), tr=7.59 min (major), 8.16 min (minor), 93.1:6.1 er.

Ethyl(R)-2-(6-((tetrahydro-2H-pyran-4-yl)methoxy)hexyl)cycloprop-2-ene-1-carboxylate(6h)

Analysis data (10 mM 5h, P411-C6):

Pdt- [Pdt]/ Avg. Avg. entries Pdt Std Pdt/Std mM [PC]/μM TTN TTN yield6h-(1) 40106 4085 9.818 7.31 1.77 4137 6h-(2) 40775 4175 9.766 7.28 1.774115 6h-(3) 39655 4125 9.613 7.16 1.77 4051 6h-(4) 38780 4188 9.260 6.901.77 3902 4051 71.6%Preparative-scale reaction:

E. coli suspension expressing P411-C6 in M9-N D-glucose in M9-N n_pro/volume/ OD₆₀₀ volume/mL [PC]/μM μmol mL [Glu]/mM 10 70.0 1.27 0.0886 3.0~10 alkyne (5h) stock in EtOH EDA stock in EtOH n_1/ n_2/ stock/Mvolume/μL mmol stock/M volume/mL mmol 1.0 400 0.40 1.0 1.20 1.20purification eluent product Ethyl acetate m[Pdt]/ n[Pdt]/ in hexanes mgmmol yield TTN (0% to 12% gradient) 81.3 0.262 66% 2960 E. colisuspension expressing P411-C6 in M9-N D-glucose in M9-N n_pro/ [Glu]/OD₆₀₀ volume/mL [PC]/μM μmol volume/mL mM 20 200 4.84 0.968 12.0 ~11alkyne (5h) stock in EtOH EDA stock in EtOH n_1/ n_2/ stock/M volume/mLmmol stock/M volume/mL mmol 2.0 1.50 3.0 2.0 4.50 9.0 purificationeluent product Ethyl acetate m[Pdt]/ n[Pdt]/ in hexanes mg mmol yieldTTN (0% to 12% gradient) 872.4 2.81 94% 2900

In the 3.0-mmol scale reaction, EDA stock solution was added in threeportions (1.5 mL each portion) every 1 hour.

¹H NMR (400 MHz, CDCl₃) δ 6.32 (dt, J=1.4 Hz, 1H), 4.13 (qd, J=7.1, 5.5Hz, 2H), 4.00-3.91 (m, 2H), 3.44-3.34 (m, 4H), 3.24 (d, J=6.6 Hz, 2H),2.49 (td, J=7.3, 1.4 Hz, 2H), 2.12 (d, J=1.5 Hz, 1H), 1.83 (ttt, J=10.5,6.7, 3.8 Hz, 1H), 1.67-1.62 (m, 2H), 1.60-1.52 (m, 4H), 1.43-1.34 (m,4H), 1.34-1.28 (m, 2H), 1.25 (t, J=7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃)δ 176.78, 115.66, 94.15, 76.12, 71.25, 67.90, 60.32, 35.61, 30.19,29.73, 29.11, 26.77, 26.02, 25.06, 19.86, 14.54. HRMS (FAB) m/z:311.2210 (M+H⁺); calc. for C₁₈H₃₁O₄: 311.2222. [α]²³ _(D)=+74.9±1.8° (c0.1, cyclohexane). HPLC Chiralpak IC column (n-hexane/isopropanol=88:12,1.0 mL/min), tr=17.27 min (major), 20.55 min (minor), 99.0:1.0 er.

Ethyl (R)-2-(6-(cyclopropylmethoxy)hexyl)cycloprop-2-ene-1-carboxylate(6i)

Analysis data (10 mM 5i, P411-C6):

Pdt- [Pdt]/ Avg. Avg. entries Pdt Std Pdt/Std mM [PC]/μM TTN TTN yield6i-(1) 26575 4144 6.413 4.44 2.01 2207 6i-(2) 26599 4150 6.409 4.43 2.012206 6i-(3) 26021 4086 6.368 4.40 2.01 2191 6i-(4) 26300 4154 6.331 4.382.01 2179 2196 44.1%Preparative-scale reaction:

E. coli suspension expressing P411-C6 in M9-N D-glucose in M9-N n_pro/OD₆₀₀ volume/mL [PC]/μM μmol volume/mL [Glu]/mM 32 25.0 6.48 0.162 1.5~11 alkyne (5i) stock in EtOH EDA stock in EtOH stock/M volume/μLn_1/mmol stock/M volume/μL n_2/mmol 1.0 360 0.36 1.0 900 0.90purification eluent product Ethyl acetate m[Pdt]/ n[Pdt]/ in hexanes mgmmol yield TTN (0% to 10% gradient) 61.8 0.232 64% 1430

¹H NMR (400 MHz, CDCl₃) δ 6.32 (dt, J=1.5 Hz, 1H), 4.12 (qd, J=7.1, 5.2Hz, 2H), 3.42 (t, J=6.7 Hz, 2H), 3.24 (d, J=6.9 Hz, 2H), 2.49 (td,J=7.3, 1.4 Hz, 2H), 2.12 (d, J=1.6 Hz, 1H), 1.62-1.54 (m, 4H), 1.41-1.34(m, 4H), 1.25 (t, J=7.2 Hz, 3H), 1.12-0.97 (m, 1H), 0.59-0.42 (m, 2H),0.28-0.09 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 176.78, 115.68, 94.13,75.74, 70.78, 60.31, 29.79, 29.11, 26.76, 26.04, 25.05, 19.86, 14.54,10.82, 3.13. HRMS (FAB) m/z: 267.1955 (M +Er); calc. for C₁₆H₂₇O₃:267.1960. [α]²³ _(D)=+43.1±1.5° (c 0.1, cyclohexane). HPLC Chiralpak ICcolumn (n-hexane/isopropanol =95:5, 1.0 mL/min), tr=14.19 min (major),15.27 min (minor), 95.8:4.2 er.

Ethyl (R or S)-2-([1,1’-bi(cyclopropan)])-2-ene-1-carboxylate (R orS-6j)

Preparative-scale reaction:

E. coli suspension D-glucose in M9-N expressing P411-C6 in M9-N volume/[Glu]/ OD₆₀₀ volume/mL [PC]/μM n_pro/μmol mL mM 20 25.0 4.47 0.112 1.2~11 alkyne (5j) stock in EtOH EDA stock in EtOH stock/ n_1/ volume/ n_2/mM volume/μL mmol stock/mM μL mmol 400 250 0.10 400 375 0.15 productpurification eluent m[Pdt]/ n[Pdt]/ Ether in pentane mg mmol yield TTN(0% to 20% gradient) 10.7 0.0703 70% 630 E. coli suspension D-glucose inM9-N expressing P411-K10 in M9-N volume/ [Glu]/ OD₆₀₀ volume/mL [PC]/μMn_pro/μmol mL mM 20 25.0 3.07 0.0767 1.2 ~11 alkyne (5j) stock in EtOHEDA stock in EtOH stock/ n_1/ volume/ n_2/ mM volume/μL mmol stock/mM μLmmol 400 250 0.10 400 375 0.15 product purification eluent m[Pdt]/n[Pdt]/ Ether in pentane mg mmol yield TTN (0% to 20% gradient) 11.20.0736 74% 960

¹H NMR (600 MHz, Acetone-d6) δ 6.47 (d, J=1.6 Hz, 1H), 4.13-4.03 (m,2H), 1.99 (d, J=1.5 Hz, 1H), 1.91 (tt, J=7.9, 4.6 Hz, 1H), 1.22 (t,J=7.1 Hz, 3H), 1.01-0.94 (m, 2H), 0.84-0.80 (m, 1H), 0.61-0.58 (m, 1H).¹³C NMR (151 MHz, acetone) δ 175.54, 117.94, 92.45, 59.99, 18.65, 14.46,6.79, 6.05, 5.78. HRMS (EI) m/z: 152.0865 (M^(+·)); calc. for C₉H₁₂O₂:152.0837. P411-C6: HPLC Chiralpak IC column (n-hexane/isopropanol=95:5,1.0 mL/min), tr=11.33 min (major), 11.79 min (minor), 93.4:6.6 er.P411-K10: HPLC Chiralpak IC column (n-hexane/isopropanol=95:5, 1.0mL/min), tr=11.33 min (minor), 11.74 min (major), 66.7:33.3 er.

Ethyl (R)-2-(4-chlorobutyl)cycloprop-2-ene-1-carboxylate (6k)

Analysis data (10 mM 5k, P411-C6):

Pdt- [Pdt]/ Avg. Avg. entries Pdt Std Pdt/Std mM [PC]/μM TTN TTN yield6k-(1) 322.4 517.1 0.623 0.56 1.82 308 6k-(2) 363.5 517.4 0.703 0.631.82 347 6k-(3) 342.8 523.4 0.655 0.59 1.82 324 326 6.0%Preparative-scale reaction:

E. coli suspension expressing P411-C6 in M9-N D-glucose in M9-N volume/n_pro/ volume/ [Glu]/ OD₆₀₀ mL [PC]/μM μmol mL mM 30 40.0 6.27 0.251 2.0~12 alkyne (5k) stock in EtOH EDA stock in EtOH stock/mM volume/μLn_1/mmol stock/mM volume/μL n_2/mmol 800 125 0.10 800 375 0.30purification eluent product Ethyl acetate m[Pdt]/ n[Pdt]/ in hexanes mgmmol yield TTN (0% to 8% gradient) 17.6 0.0868 87% 340

¹H NMR (400 MHz, CDCl₃) δ 6.37 (s, 1H), 4.12 (dd, J=7.1, 4.2 Hz, 2H),3.54 (t, J=6.5 Hz, 2H), 2.54 (t, J=7.0 Hz, 2H), 2.13 (s, 1H), 1.91-1.80(m, 2H), 1.80-1.70 (m, 2H), 1.24 (t, J=7.1 Hz, 3H). ¹³C NMR (101 MHz,CDCl₃) δ 176.54, 114.98, 94.94, 60.37, 44.67, 31.89, 24.39, 24.12,19.83, 14.49. HRMS (FAB) m/z: 203.0846 (M+H⁺); calc. for C₁₀H₁₆ClO₂:203.0839. [α]²³ _(D)=+25.7±1.6° (c 0.1, cyclohexane). HPLC Chiralpak ICcolumn (n-hexane/isopropanol=95:5, 1.0 mL/min), tr=11.00 min (major),12.62 min (minor), 94.2:5.8 er.

Ethyl(R)-2-(6-((trans-4-(methoxymethyl)cyclohexyl)methoxy)hexyl)cycloprop-2-ene-1-carboxylate(61)

Analysis data (10 mM 51, P411-C6):

Pdt- [Pdt]/ Avg. Avg. entries Pdt Std Pdt/Std mM [PC]/μM TTN TTN yield6l-(1) 14861 4259 3.489 2.41 1.93 1252 6l-(2) 13735 4274 3.214 2.22 1.931153 6l-(3) 16271 4222 3.854 2.66 1.93 1383 6l-(4) 13204 4323 3.054 2.111.93 1096 1221 23.5%Preparative-scale reaction:

E. coli suspension expressing P411-C6 in M9-N D-glucose in M9-N n_pro/volume/ OD₆₀₀ volume/mL [PC]/μM μmol mL [Glu]/mM 10 70.0 1.27 0.0886 3.0~10 alkyne (5l) stock in EtOH EDA stock in EtOH stock/M volume/μLn_1/mmol stock/M volume/μL n_2/mmol 1.0 180 0.18 1.0 540 0.54purification eluent product Ethyl acetate m[Pdt]/ n[Pdt]/ in hexanes mgmmol yield TTN (0% to 15% gradient) 22.1 0.0627 35% 710

The cell suspension (OD₆₀₀=10) was added in two portions (35 mL eachportion) every 2 hours.

¹H NMR (400 MHz, CDCl₃) δ 6.32 (dt, J=1.4 Hz, 1H), 4.12 (qd, J=7.1, 5.4Hz, 2H), 3.37 (t, J=6.6 Hz, 2H), 3.32 (s, 3H), 3.21-3.17 (m, 4H), 2.49(td, J=7.3, 1.4 Hz, 2H), 2.12 (d, J=1.6 Hz, 1H), 1.87-1.74 (m, 4H),1.63-1.52 (m, 6H), 1.37 (tt, J=6.3, 3.3 Hz, 4H), 1.25 (t, J=7.1 Hz, 3H),0.99-0.86 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) δ 176.79, 115.69, 94.12,78.93, 76.90, 71.17, 60.31, 58.98, 38.37 (two carbons), 29.74, 29.65,29.55, 29.11, 26.77, 26.03, 25.06, 19.86, 14.54. HRMS (FAB) m/z:353.2699 (M+H⁺); calc. for C₂₁H₃₇O₄: 353.2692. [α]²³ _(D)=+62.6±2.7° (c0.1, cyclohexane). HPLC Chiralpak IC column (n-hexane/isopropanol=93:7,1.0 mL/min), tr=10.07 min (major), 11.44 min (minor), 96.4:3.6 er.

Ethyl (R)-2-(6-hydroxyhexyl)cycloprop-2-ene-1-carboxylate (6m)

Preparative-scale reaction:

E. coli suspension expressing P411-C6 in M9-N D-glucose in M9-N n_pro/[Glu]/ OD₆₀₀ volume/mL [PC]/μM μmol volume/mL mM 30 40.0 6.80 0.272 1.5~12 alkyne (5m) stock in EtOH EDA stock in EtOH volume/ n_2/ stock/M μLn_1/mmol stock/M volume/μL mmol 1.0 250 0.25 2.0 750 1.50 purificationeluent product Ethyl acetate m[Pdt]/ n[Pdt]/ in hexanes mg mmol yieldTTN (0% to 25% gradient) 38.2 0.193 80% 710

¹El NMR (400 MHz, Acetone-d6) δ 6.56 (dt, J=1.5 Hz, 1H), 4.05 (qd,J=7.1, 2.5 Hz, 2H), 3.52 (t, J=6.2 Hz, 2H), 2.50 (tt, J=7.1, 1.6 Hz,2H), 2.03 (d, J=1.6 Hz, 1H), 1.63-1.56 (m, 2H), 1.55-1.49 (m, 2H),1.48-1.40 (m, 2H), 1.19 (t, J=7.1 Hz, 3H) (0-H proton is not resolved).¹³C NMR (101 MHz, Acetone-d6) δ 176.12, 116.11, 94.99, 62.12, 60.23,33.34, 27.33, 26.16, 25.47, 19.98, 14.72. HRMS (FAB) m/z: 199.1342(M+H⁺); calc. for C₁₁H₁₉O₃: 199.1334. [α]²³ _(D)=+80.1±3.4° (c 0.1,ethyl acetate). HPLC Chiralpak IC column (n-hexane/isopropanol=88:12,1.0 mL/min), tr=15.76 min (major), 16.86 min (minor), 96.0:4.0 er.

During the test of analytical reactions, aliphatic alkynes 5n-5q alsogave a fair amount of cyclopropene products, which were detected byGC-MS. Making the racemic standards with Rh₂(OAc)₄ was problematic,resulting in no product formation or products with other impurities.

Example 15 Chemical Elaboration of Cyclopropene Products

Cyclopropenes are used as synthetic building blocks (10, ⁴⁷),bio-orthogonal imaging precursors (⁴⁸), and monomers in polymersynthesis (⁴⁹). The ability to construct these motifs using bacteria atscale allows for the further exploration of their potential utility indiverse fields. Two simple transformations of cyclopropenes were carriedout to build a multi-substituted cyclopropane 7a and a fused ringsystem, bicyclo[4.1.0]heptene 7b (FIG. 3C), both of which aresubstructures common in pharmaceutical candidates and bioactive naturalproducts (27).

Diethyl(2R,4R)-1-(4-(1-((4-acetamidophenyl)sulfonyl)-1H-1,2,3-triazol-4-yl)phenyl)bicyclo[1.1.0]butane-2,4-dicarboxylate (4a)

Bicyclobutane 21 (41.6 mg, 0.139 mmol) was dissolved in toluene (3 mL)in a 20 mL vial. Copper(I) thiophene-2-carboxylate (CuTc, 3.2 mg, 12 mol%) was added. The mixture was kept stirring at 0° C. A suspension of4-acetamidobenzenesulfonyl azide (40.2 mg, 0.167 mmol) in toluene (5 mL)was added dropwise over 1 h. The resulting mixture was then allowed towarm to room temperature and stirred for 15 h. The reaction was dilutedwith ethyl acetate (15 mL), washed with NH₄Cl/NH₃ (2:1, 20 mL, aq.), andbrine (50 mL), dried over MgSO₄, and then concentrated under reducedpressure. The crude product was purified by silica column chromatographywith hexane/ethyl acetate (4:1 to 1:3 gradient) to afford 4a (69.2 mg,0.129 mmol, 93%). ¹H NMR (400 MHz, CDCl₃) δ 8.32 (s, 1H), 8.10 (s, 1H),8.00 (d, J=9.0 Hz, 2H), 7.74 (dd, J=12.8, 8.7 Hz, 4H), 7.34 (d, J=8.6Hz, 2H), 4.15 (dq, J=7.1, 3.5 Hz, 2H), 4.08 (q, J=7.1 Hz, 2H), 3.28 (d,J=0.6 Hz, 1H), 3.19 (dd, J=3.1, 0.6 Hz, 1H), 3.14 (d, J=3.1 Hz, 1H),2.20 (s, 3H), 1.27 (t, J=7.2 Hz, 3H), 1.15 (t, J=7.1 Hz, 3H). ¹³C NMR(101 MHz, CDCl₃) δ 170.14, 169.15, 167.88, 147.11, 145.13, 134.03,130.40, 129.53, 128.96, 127.92, 126.19, 119.62, 119.22, 61.17, 61.13,44.75, 42.63, 27.84, 24.93, 21.39, 14.35, 14.21. HRMS (FAB) m/z:539.1605 (M+H⁺); calc. for C₂₆H₂₇O₇SN₄: 539.1600. [α]²³ _(D)=−81.5±2.1°(c 0.1, ethyl acetate).

Diethyl(2R,4R)-1-(4-(14(S)-1-methoxy-1-oxo-3-phenylpropan-2-yl)-1H-1,2,3-triazol-4-yl)phenyl)bicyclo[1.1.0]butane-2,4-dicarboxylate(4b)

Sodium azide (7.02 g, 108 mmol) was dissolved in a mixture of water (30mL) and DCM (15 mL) at 0° C. Triflic anhydride (Tf₂O, 3 mL, 18 mmol) wasadded dropwise over 10 min. The reaction was then stirred for 2 h. Thereaction was diluted with DCM (15 mL), washed by NaHCO₃ (50 mL, sat.aq.), and brine (50 mL), and dried over Na₂SO₄. A solution of triflicazide (TfN₃, ˜15 mmol) in DCM was used for the next step.

Methyl L-phenylalaninate hydrochloride (1.73 g, 8.0 mmol), was dissolvedin MeOH (16 mL). A aqueous solution of CuSO₄ (1.28 mL, 10 g/L, 1 mol %)and N,N-diisopropylethylamine (DIPEA, 2.09 mL, 12.0 mmol) were added.The resulting solution was stirred for 30 min before the dropwiseaddition of triflic azide (TfN₃, ˜15 mmol in DCM) over 20 min. Thereaction was stirred for 15 h. The reaction mixture was diluted with DCM(40 mL), washed by water (50 mL), HCl (50 mL, 5%, aq.), NaHCO₃ (50 mL,sat. aq.), and brine (50 mL), and dried over Na₂SO₄. After evaporationof solvent under reduced pressure, the product mixture was purified bysilica column chromatography with hexane/ethyl acetate (1:0 to 12:1gradient) to afford methyl (S)-2-azido-3-phenylpropanoate (1.65 g, 8.0mmol, quantitative).

Bicyclobutane 2l (49.5 mg, 0.166 mmol) was dissolved in THF/H₂O(1:1, 1.6mL) in a 20 mL vial. A aqueous solution of CuSO₄ (17 μL, 1N, 10 mol %)and Cu powder (10.6 mg, 0.166 mmol) were added. The mixture was keptstirring at 0° C. A solution of methyl (S)-2-azido-3-phenylpropanoate(50.1 mg, 0.249 mmol) in THF (0.5 mL) was added dropwise over 5 min. Theresulting mixture was then allowed to warm to room temperature andstirred for 24 h. The reaction was diluted with DCM (15 mL), washed withNH₄Cl (10 mL, aq. sat.), and brine (30 mL), dried over MgSO₄, and thenconcentrated under reduced pressure. The crude product was purified bysilica column chromatography with hexane/ethyl acetate (8:1 to 2:1gradient) to afford 4b (82.5 mg, 0.164 mmol, 99%) as one majordiastereomer determined by ¹H NMR. ¹H NMR (400 MHz, CDCl₃) δ 7.80 (s,1H), 7.78-7.69 (m, 2H), 7.37-7.30 (m, 2H), 7.28-7.20 (m, 3H), 7.08-7.03(m, 2H), 5.63 (dd, J=8.1, 6.7 Hz, 1H), 4.15 (qd, J=7.1, 2.6 Hz, 2H),4.08 (q, J=7.1 Hz, 2H), 3.77 (s, 3H), 3.52 (dd, J=7.4, 3.2 Hz, 2H), 3.28(d, J=0.5 Hz, 1H), 3.17 (dd, J=3.1, 0.6 Hz, 1H), 3.13 (d, J=3.1 Hz, 1H),1.27 (t, J=7.1 Hz, 3H), 1.15 (t, J=7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃)δ 170.24, 168.84, 167.87, 147.40, 134.77, 132.77, 129.67, 129.08,128.98, 128.84, 127.77, 125.80, 119.75, 64.17, 61.05, 61.01, 53.26,44.71, 42.64, 39.12, 27.99, 20.89, 14.36, 14.22. HRMS (FAB) m/z:504.2148 (M+H⁺); calc. for C₂₈H₃₀O₆N₃: 504.2135. [α]²³ _(D)=−278.7±4.0°(c 0.0625, ethyl acetate).

A diastereomer of 4b was prepared through the same procedure usingD-azido-phenylalanine. It was confirmed that the pair of diastereomerscan be resolved on ¹H NMR at the α-C—H position of phenylalanine(splitting peaks: D-phenylalanine: 5.6410, 5.6242, 5.6208, 5.6041;L-phenylalanine: 5.6462, 5.6296, 5.6261, 5.6093; FWHM (full width athalf maxima)=0.0030 ppm; Δf1=0.0053 ppm, close to baseline separation).

Diethyl(2R,4R)-1-(4-((((R)-3,3,3-Trifluoro-2-methoxy-2-phenylpropanoyl)oxy)methyl)phenyl)bicyclo[1.1.0]butane-2,4-dicarboxylate (4c)

Bicyclobutane 2m (16.1 mg, 0.053 mmol) and(R)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoic acid (R-Mosher's acid,30.4 mg, 0.13 mmol) were dissolved in dry DCM (2 mL) in a 10 mL vial.1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl, 24.9mg, 0.13 mmol), triethylamine (TEA, 18 μL, 0.13 mmol) and4-dimethylaminopyridine (DMAP, 2.0 mg, 0.015 mmol) were added to thesolution. The reaction mixture was then stirred at room temperature for16 h. The reaction was diluted with DCM (5 mL), washed with water (10mL), and brine (10 mL), dried over MgSO₄, and then concentrated underreduced pressure. The crude product was purified by silica columnchromatography with hexane/ethyl acetate (1:0 to 4:1 gradient) to afford4c (25.2 mg, 0.048 mmol, 91%) as one major diastereomer determined by ¹HNMR. ¹H NMR (400 MHz, CDCl₃) δ 7.48-7.41 (m, 2H), 7.41-7.32 (m, 3H),7.27 (s, 4H), 5.33 (d, J=12.3 Hz, 1H), 5.29 (d, J=12.2 Hz, 1H), 4.15(qd, J=7.2, 2.6 Hz, 2H), 4.07 (qd, J=7.2, 2.0 Hz, 2H), 3.51 (q,J_(H-F)=1.2 Hz, 3H), 3.26 (d, J=0.6 Hz, 1H), 3.14 (dd, J=3.1, 0.6 Hz,1H), 3.11 (d, J=3.1 Hz, 1H), 1.27 (t, J=7.1 Hz, 3H), 1.13 (t, J=7.1 Hz,3H). ¹³C NMR (101 MHz, CDCl₃) δ 170.01, 167.67, 166.43, 133.67, 133.32,132.11, 129.64, 128.54, 128.53, 128.42, 127.54, 127.28, 127.27, 124.68,121.81, 118.94, 84.75, 67.60, 60.96, 60.88, 55.54, 44.61, 42.44, 27.66,20.85, 14.23, 14.03 (CF3, 127.54, 124.68, 121.81, 118.94, Jc-F =288.8Hz;). HRMS (FAB) m/z: 519.1651 ((M+H⁺)−H₂); calc. for C₂₇H₂₆O₇F₃:519.1631. [α]²³ _(D)=−43.2±2.4° (c 0.1, ethyl acetate).

A diastereomer of 4c was prepared through the same procedure usingS-Mosher's acid. It was confirmed that the pair of diastereomers can beresolved on ¹H NMR at one benzylic C—H position and ¹⁹F NMR, albeit notperfectly (splitting peaks on ¹H NMR: R-Mosher's acid: 5.3027, 5.2721;S-Mosher's acid: 5.3044, 5.2737; FWHM=0.0024-0.0029 ppm ; Δf1=0.0017ppm; chemical shift on ¹⁹F NMR: R-Mosher's acid: −71.665; S-Mosher'sacid: −71.654; FWHM=0.015 ppm ; Δf1=0.011 ppm).

((1R,2R)-1-(4-methoxyphenyl)bicyclo[1.1.0]butane-2,4-diyl)dimethanol(4d)

Bicyclobutane 2k (101.3 mg, 0.33 mmol) was dissolved in anhydrous ether(10 mL) in a 50 mL flask. Dry methanol (67 μL, 1.67 mmol) was added tothe solution, followed by the addition of lithium borohydride (LiBH₄, 2Min THF, 0.84 mL, 1.67 mmol). The reaction mixture was then heated up toa refluxing temperature, 45° C., for 1 h, before ethyl acetate (0.5 mL)was added and the mixture was stirred at room temperature for 30 min.Then the reaction was quenched with NH₄Cl (10 mL, sat. aq.) and dilutedwith water (10 mL). The product was extracted with ethyl acetate (20mL×6). The combined organic layer was dried over MgSO₄, and thenconcentrated under reduced pressure. White solid product crashed outduring the removal of solvent. Collecting the solid product andrecrystallization with acetone/hexane system afforded the diol 4d (52.2mg, 0.237 mmol, 71%). ¹H NMR (400 MHz, Acetone-d6) δ 7.31 (d, J=8.9 Hz,2H), 6.85 (d, J=8.8 Hz, 2H), 3.78 (s, 3H), 3.71-3.65 (m, 1H), 3.65-3.61(m, 1H), 3.57-3.51 (m, 1H), 3.49-3.43 (m, 2H), 2.28 (t, J=5.8 Hz, 1H),1.92 (dd, J=3.6, 1.0 Hz, 1H) (0-H protons are not resolved). ¹³C NMR(101 MHz, Acetone) δ 158.07, 129.14, 129.08, 113.58, 60.27, 60.14(splitting), 55.88, 55.75 (splitting), 54.56, 49.65, 49.61 (splitting),46.08, 46.04 (splitting), 21.53, 16.55. (Note: the diol product mighthave two rotating conformations, resulting in splitting of 4 carbonpeaks.) HRMS (FAB) m/z: 221.1168 (M+H⁺); calc. for C₁₃H₁₇O₃: 221.1178.[α]²³ _(D)=−50.8±1.2° (c 0.1, acetone).

Crystal structure information for 3d:

Identification code p17505 Empirical formula C13 H16 O3 Formula weight220.26 Temperature 175 K Wavelength 1.54178 Å Crystal system MonoclinicSpace group P 1 21 1 Unit cell dimensions a = 5.3738(7) Å a = 90° b =7.6719(8) Å b = 98.413(9)° c = 13.7357(19) Å g = 90° Volume 560.19(12)Å³ Z 2 Density (calculated) 1.306 Mg/m³ Absorption coefficient 0.747mm⁻¹ F(000) 236 Crystal size 0.22 × 0.18 × 0.06 mm³ Theta range for datacollection 3.252 to 79.483°. Index ranges −6 <= h <= 6, −9 <= k <= 9,−16 <= l <= 17 Reflections collected 17841 Independent reflections 2338[R(int) = 0.0437] Completeness to theta = 67.679° 99.9% Absorptioncorrection Semi-empirical from equivalents Max. and min. transmission1.0000 and 0.8812 Refinement method Full-matrix least-squares on F²Data/restraints/parameters 2338/1/148 Goodness-of-fit on F² 1.111 FinalR indices [I > 2sigma(I)] R1 = 0.0471, wR2 = 0.1225 R indices (all data)R1 = 0.0482, wR2 = 0.1232 Absolute structure parameter 0.15(7)Extinction coefficient n/a Largest diff. peak and hole 0.337 and −0.270e · Å⁻³

Ethyl(1R,2R)-4-(hydroxymethyl)-1-(4-methoxyphenyl)bicyclo[1.1.0]butane-2-carboxylate(4e)

Mono-alcohol 4e was obtained from the reduction of bicyclobutane 2k.Collecting the mother liquor after recrystallization, evaporation oforganic solvent followed by purification by silica column chromatographywith hexane/ethyl acetate (5:1 to 3:1 gradient) afforded 4e (14.6 mg,0.056 mmol, 17%) with ˜7:1 r.r. determined by ¹H NMR. ¹H NMR (400 MHz,Acetonitrile-d₃) δ 7.20 (d, J=8.8 Hz, 2H), 6.88 (d, J=8.8 Hz, 2H), 4.05(qd, J=7.1, 0.5 Hz, 2H), 3.78 (s, 3H), 3.43 (t, J=6.3 Hz, 2H), 2.75(tdd, J=7.0, 3.3, 0.7 Hz, 1H), 2.70 (t, J=5.9 Hz, 1H), 2.68 (s, 1H),2.64 (d, J=3.3 Hz, 1H), 1.14 (t, J=7.1 Hz, 3H). ¹³C NMR (101 MHz, CD3CN)δ 168.54, 159.04, 129.70, 127.38, 114.20, 60.92, 56.42, 55.44, 47.23,42.52, 24.86, 18.43, 13.99. MS (EI) m/z: 262 (M^(+·)); calc. forC₁₅H₁₈O₄: 262.

(2-(4-Methoxyphenyl)cyclobut-1-ene-1,3-diyl)dimethanol (4f)

Bicyclobutane 2k (54.7 mg, 0.18 mmol) was dissolved in anhydrous ether(5 mL) in a 20 mL vial and then cooled down to −78° C. Lithium aluminumhydride (LAH, 13.7 mg, 0.36 mmol) was slowly added to the solution inportions. The reaction mixture was slowly warmed up to −20° C. over 2 h,before ethyl acetate (0.5 mL) was added and the mixture was stirred at−20° C. for another 30 min. Then the reaction was quenched with NH₄Cl(10 mL, sat. aq.) and diluted with water (10 mL). The product wasextracted with ethyl acetate (20 mL×3). The combined organic layer wasdried over MgSO₄, and then concentrated under reduced pressure. Thecrude product was purified by silica column chromatography withhexane/ethyl acetate (2:1 to 1:2 gradient) to afford 4f (18.1 mg, 0.082mmol, 46%). ¹H NMR (400 MHz, Acetonitrile-d3) δ 7.34 (d, J=8.8 Hz, 2H),6.92 (d, J=8.9 Hz, 2H), 4.30 (d, J=3.1 Hz, 2H), 3.80 (s, 5H), 3.57 (ddd,J=11.2, 7.2, 4.3 Hz, 1H), 3.21-3.12 (m, 1H), 2.95 (t, J=5.7 Hz, 1H),2.70 (t, J=5.5 Hz, 1H), 2.59 (dd, J=13.7, 4.6 Hz, 1H). ¹³C NMR (101 MHz,CD3CN) δ 159.59, 140.27, 140.19, 128.91, 128.55, 114.63, 65.02, 59.23,55.79, 42.11, 31.40. MS (EI) m/z: 220 (M⁺); calc. for C₁₃H₁₆O₃: 220.

Diethyl(2R,4R)-1-(4-(((4-(4,5,6,7-Tetrachloro-1,3-dioxoisoindolin-2-yl)benzoyl)oxy)methyl)phenyl)bicyclo[1.1.0]butane-2,4-dicarboxylate (4g)

Tetrachlorophthalic anhydride (14.30 g, 50.0 mmol) and 4-aminobenzoicacid (6.86 g, 50.0 mmol) were dissolved in PhMe (100 mL) to form asuspension. The mixture was heated up to 110° C. and stirred vigorously,before the addition of TEA (0.35 mL, 5 mol %). The reaction turned clearand then white product started to crash out. The reaction was stirred atthe refluxing temperature for 1 h, and then slowly cooled to roomtemperature. The product4-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl)benzoic acid (20.05 g,49.5 mmol, 99%) was collected by filtration.

4-(4,5,6,7-Tetrachloro-1,3-dioxoisoindolin-2-yl)benzoic acid (40.5 mg,0.1 mmol) and DMF (3 drops, cat.) was dissolved in dry DCM (8 mL) andSOCl₂ (2 mL). The mixture was heated and stirred at a refluxingtemperature, 48° C. for 4 h. After completely removing organic solventand excess SOCl₂ under reduced pressure, the resulting crude product4-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl)benzoyl chloride wasdissolved in dry DCM (3 mL) and used directly for next step.

Bicyclobutane 2m (15.2 mg, 0.050 mmol) and TEA (14 μL, 0.10 mmol) weredissolved in dry DCM (2 mL) in a 10 mL vial. The solution of preparedbenzoyl chloride in DCM was added to the solution. The reaction mixturewas then stirred at room temperature for 12 h. The reaction was dilutedwith DCM (5 mL), washed with water (10 mL), and brine (10 mL), driedover MgSO₄, and then concentrated under reduced pressure. The crudeproduct was purified by silica column chromatography with hexane/ethylacetate (1:0 to 4:1 gradient) to afford 4g (30.0 mg, 0.0434 mmol, 87%)as a white solid. ¹H NMR (400 MHz, Acetonitrile-d₃) δ 8.21 (d, J=8.6 Hz,2H), 7.60 (d, J=8.7 Hz, 2H), 7.47 (d, J=8.3 Hz, 2H), 7.34 (d, J=8.2 Hz,2H), 5.38 (s, 2H), 4.09 (dq, J=14.2, 7.1 Hz, 4H), 3.30 (dd, J=3.1, 0.6Hz, 1H), 3.16 (d, J=3.1 Hz, 1H), 3.07 (d, J=0.5 Hz, 1H), 1.23 (t, J=7.1Hz, 3H), 1.15 (t, J=7.1 Hz, 3H). ¹³C NMR (101 MHz, CD3CN) δ 170.14,167.93, 165.82, 163.12 (two carbons), 140.42, 136.05, 135.95, 133.64,130.76, 130.67, 130.00, 128.81, 128.72, 128.55, 127.57, 66.99, 61.30,61.25, 45.10, 42.39, 27.43, 21.38, 14.04, 13.92. HRMS (ESI) m/z:712.0082 (M+H⁺); calc. for C₃₂H₂₃C₁₄NO₈Na: 712.0075. [α]²³_(D)=−48.5±1.6° (c 0.1, acetone).

Ethyl (1R,2R,3S)-3-allyl-2-methyl-2-phenethylcyclopropane-1-carboxylate(7a)

Cyclopropene (R)-6a (54.0 mg, 0.25 mmol) and CuI (5.0 mg, 0.026 mmol, 10mol %) were dissolved in anhydrous ether (3 mL) to form a suspension ina 25 mL flask. Then the flask was charged with argon and cooled to −78°C. Methylmagnesium bromide (0.75 mL, 1M in ether, diluted from 3Msolution in ether) was added dropwise to the reaction mixture over 10min. The reaction was slowly warmed to -40° C. over 30 min and held atthis temperature for another 1 h. A solution of allyl bromide (43 μL,0.50 mmol) in ether (1 mL) was then added to the reaction dropwise over5 min. The reaction was stirred at −40° C. for 1 h, before it wasquenched with NH₄Cl/NH₃ (2:1, aq., 5 mL) at −20° C. The aqueous layerwas extracted twice with ether. The combined organic layers were washedwith water (10 mL), and brine (10 mL), dried over MgSO₄, and thenconcentrated under reduced pressure. The crude product was purified bysilica column chromatography with hexane/ethyl acetate (1:0 to 50:1gradient) to afford 7a (48.0 mg, 0.176 mmol, 71%) as a singlediastereomer. ¹H NMR (400 MHz, CDCl₃) δ 7.23-7.17 (m, 2H), 7.13-7.07 (m,3H), 5.73 (ddt, J=17.3, 10.2, 6.2 Hz, 1H), 4.97 (dq, J=17.2, 1.7 Hz,1H), 4.89 (ddt, J=10.2, 2.0, 1.4 Hz, 1H), 4.07-3.96 (m, 2H), 2.68-2.60(m, 2H), 2.33 (ddt, J=7.6, 6.2, 1.5 Hz, 2H), 1.57-1.51 (m, 2H), 1.40 (d,J=8.9 Hz, 1H), 1.23 (s, 3H), 1.18 (t, J=7.1 Hz, 3H), 1.09 (dt, J=8.8,7.4 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 171.68, 142.04, 137.72, 128.38,128.33, 125.81, 114.58, 59.80, 45.06, 32.87, 31.77, 29.13, 27.89, 27.34,14.39, 11.53. HRMS (FAB) m/z: 273.1849 (M+H⁺); calc. for C₁₈H₂₅O₂:273.1855. [α]²³ _(D)=−61.3±1.7° (c 0.1, ethyl acetate).

Ethyl(1R,6R,7R)-3,4-dimethyl-1-(6-((tetrahydro-2H-pyran-4-yl)methoxy)hexyl)bicyclo[4.1.0]hept-3-ene-7-carboxylate (7b)

Cyclopropene (R)-6g (65.6 mg, 0.211 mmol) was dissolved2,3-dimethylbutadiene (0.5 mL). Then the reaction was heated and stirredat 80° C. in a sealed tube for 23 h. After cooling to room temperature,the resulting mixture was concentrated under reduced pressure. The crudeproduct was purified by silica column chromatography with hexane/ethylacetate (1:0 to 6:1 gradient) to afford 7b (81.1 mg, 0.207 mmol, 98%) asa single diastereomer. ¹H NMR (400 MHz, CDCl₃) δ 4.09 (qd, J=7.1, 0.4Hz, 2H), 4.01-3.90 (m, 2H), 3.45-3.32 (m, 4H), 3.24 (d, J=6.6 Hz, 2H),2.43-2.29 (m, 1H), 2.30-2.09 (m, 3H), 1.90-1.75 (m, 1H), 1.67-1.60 (m,4H), 1.58-1.50 (m, 10H), 1.46-1.38 (m, 1H), 1.36-1.26 (m, 5H), 1.26-1.15(m, 5H). ¹³C NMR (101 MHz, CDCl₃) δ 173.76, 122.00, 121.77, 76.09,71.37, 67.91, 60.21, 35.85, 35.60, 32.93, 32.16, 30.85, 30.20, 29.80,29.69, 27.92, 26.89, 26.86, 26.33, 19.30, 19.09, 14.55. HRMS (FAB) m/z:393.2992 (M+H⁺); calc. for C₂₄H₄₁O₄: 393.3005. [α]²³ _(D)=−48.2±1.1° (c0.1, ethyl acetate).

A biocatalytic platform has been developed for the construction ofhighly strained bicyclobutanes and cyclopropenes through directedevolution of a serine-ligated cytochrome P450 (P411) enzyme. That theprotein could be quickly adapted to produce these highly strainedstructures (2-6 mutational steps) highlights the evolvability of theP411 scaffold and its potential to direct the construction of complexmotifs. The protein enabled the desired transformations throughactivation of iron-carbenoid for carbene addition to alkynes,stabilization of the reactive cyclopropene intermediate (inbicyclobutane formation), and precise stereocontrol of the carbenetransfer processes. Biotransformations with the evolved enzymes have asurprisingly broad substrate scope with high reactivity and selectivity,providing a route to more than 25 products in preparative scale. Thisbiocatalytic system grants facile access to versatile moleculararchitectures rarely seen in nature, expanding the set of chemicalstructures available to biological systems.

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Although the foregoing has been described in some detail by way ofillustration and example for purposes of clarity and understanding, oneof skill in the art will appreciate that certain changes andmodifications can be practiced within the scope of the appended claims.In addition, each reference provided herein is incorporated by referencein its entirety to the same extent as if each reference was individuallyincorporated by reference.

1. A method for preparing a product containing a cyclopropene moiety ora bicyclobutane moiety, the method comprising combining an alkyne and acarbene precursor in the presence of a heme protein or a variant thereofunder conditions sufficient to form the product containing thecyclopropene moiety or the bicyclobutane moiety.
 2. The method of claim1, wherein the heme protein variant comprises a mutation at an axialheme coordination residue.
 3. The method of claim 1, wherein the hemeprotein is a cytochrome P450.
 4. The method of claim 3, wherein thecytochrome P450 is Bacillus megaterium cytochrome P450_(BM3) or avariant thereof.
 5. The method of claim 4, wherein the P450_(BM3)comprises one or more amino acid mutations at positions V78, F87, P142,T175, A184, 5226, H236, E252, 1263, T268, A290, A328, L353, 1366, C400,T438, and E442 relative to the amino acid sequence set forth in SEQ IDNO:1.
 6. The method of claim 1, wherein the heme protein is a cytochromeP450_(BM3) variant comprising the heme domain amino acid sequence setforth in SEQ ID NO:2, and wherein the amino acid sequence optionallycomprises an amino acid mutation at one or more positions selected fromS72, A78, A87, L188, F261, T269, T327, V328, A330, T436, and L437. 7-25.(canceled)
 26. The method of claim 6, wherein the product comprises acyclopropene. 27-31. canceled
 32. The method of claim 26, wherein thecarbene precursor is a diazo compound. 33-34. (canceled)
 35. The methodof claim 26, wherein the product is a cyclopropene according to FormulaIII:

wherein R¹⁰ is selected from the group consisting of H and CR¹R²; eachR¹ is independently selected from the group consisting of H, C₁₋₁₈alkyl, C₁₋₁₈ haloalkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, (C₃₋₁₀cycloalkyl)-L¹-, (C₆₋₁₀ aryl)-L¹-, (5- to 10-membered heteroaryl)-L¹-,(5- to 10-membered heterocyclyl)-L¹-, cyano, halo, nitro, N(R⁸)₂,B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷, C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂,C(O)NR⁷OR⁸, C(O)C(O)OR⁷, and P(O)(OR⁷)₂; C₁₋₁₈ alkyl, C₃₋₁₀ cycloalkyl,C₆₋₁₀ aryl, 5- to 10-membered heteroaryl, and 5- to 10-memberedheterocyclyl in R¹ are optionally and independently substituted with oneor more R^(1a); each R^(1a) is independently selected from the groupconsisting of halogen, cyano, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆heteroalkyl, C₂₋₆ alkynyl, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, and 5- to10-membered heterocyclyl; L¹ is selected from the group consisting of abond and C₁₋₂₀ alkylene; when L¹ is C₂₋₂₀ alkylene, one or morenon-adjacent CH₂ groups are optionally and independently replaced withO, S, or NH; when L¹ is C₃₋₂₀ alkylene, one or more pairs of adjacentCH₂ groups are optionally and independently replaced with C(O)O orC(O)NH; each R² is independently selected from the group consisting ofH, C₁₋₁₈ alkyl, C₁₋₁₈ haloalkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, (C₃₋₁₀cycloalkyl)-L²-, (C₆₋₁₀ aryl)-L²-, (5- to 10-membered heteroaryl) L²-,(5- to 10-membered heterocyclyl)-L²-, cyano, halo, nitro, N(R⁸)₂,B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷, C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂,C(O)NR⁷OR⁸, C(O)C(O)OR⁷, and P(O)(OR⁷)₂; C₁₋₁₈ alkyl, C₃₋₁₀ cycloalkyl,C₆₋₁₀ aryl, 5- to 10-membered heteroaryl, and 5- to 10-memberedheterocyclyl in R² are optionally and independently substituted with oneor more R^(2a); each R^(2a)a is independently selected from the groupconsisting of halogen, cyano, —OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆heteroalkyl, C₂₋₆ alkynyl, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, and 5- to10-membered heterocyclyl; L² is selected from the group consisting of abond and C₁₋₂₀ alkylene; when L² is C₂₋₂₀ alkylene, one or morenon-adjacent CH₂ groups are optionally and independently replaced withO, S, or NH; when L² is C₃₋₂₀ alkylene, one or more pairs of adjacentCH₂ groups are optionally and independently replaced with C(O)O orC(O)NH; and R³ and R⁴ are independently selected from the groupconsisting of H, optionally substituted C₁₋₁₈ alkyl, optionallysubstituted C₁₋₁₈ alkoxy, C₁₋₁₈ haloalkyl (e.g., C₁₋₁₈ polyfluoroalkyl),C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl,optionally substituted 5- to 10-membered heteroaryl, optionallysubstituted 5- to 10-membered heterocyclyl, cyano, halo, nitro, N(R⁸)₂,B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷, C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂,C(O)NR⁷OR⁸, C(O)C(O)OR⁷ and P(O)(OR⁷)₂; and each R⁷, R⁸, and R⁹ isindependently selected from the group consisting of H, optionallysubstituted C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, optionallysubstituted C₆₋₁₀ aryl, optionally substituted 5- to 10-memberedheteroaryl, and optionally substituted 5- to 10-membered heterocyclyl.36-39. (canceled)
 40. The method of claim 1, wherein the heme protein isa cytochrome P450_(BM3) variant comprising the heme domain amino acidsequence set forth in SEQ ID NO:3, and wherein the amino acid sequenceoptionally comprises an amino acid mutation at one or both of positionsV78 and S438. 41-45 (canceled)
 46. The method of claim 40, wherein theproduct comprises a bicyclobutane. 47-50. (canceled)
 51. The method ofclaim 46, wherein the carbene precursor is a diazo compound. 52-53.(canceled)
 54. The method of claim 46, wherein the product is abicyclobutane according to Formula VI:

wherein R¹¹ is selected from the group consisting of H, C₁₋₁₈ alkyl,C₁₋₁₈ haloalkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, (C₃₋₁₀ cycloalkyl)-L¹¹-,(C₆₋₁₀ aryl)-L¹¹-, (5- to 10-membered heteroaryl)-L¹¹-, (5- to10-membered heterocyclyl)-L¹¹-, cyano, halo, nitro, N(R¹⁸)₂, B(R¹⁹)₂,Si(R¹⁹)₃, C(O)OR¹⁷, C(O)SR¹⁷, C(O)N(R¹⁷)₂, C(O)R¹⁷, C(O)ON(R¹⁷)₂,C(O)NR¹⁷OR¹⁸, C(O)C(O)OR¹⁷, and P(O)(OR¹⁷)₂; C₁₋₁₈ alkyl, C₃₋₁₀cycloalkyl, C₆₋₁₀ aryl, 5- to 10-membered heteroaryl, and 5- to10-membered heterocyclyl in R¹¹ are optionally and independentlysubstituted with one or more R^(11a); each R^(11a) is independentlyselected from the group consisting of halogen, cyano, —OH, C₁₋₆ alkyl,C₁₋₆ alkoxy, C₁₋₆ heteroalkyl, C₂₋₆ alkynyl, C₃₋₁₀ cycloalkyl, C₆₋₁₀aryl, and 5- to 10-membered heterocyclyl; L¹¹ is selected from the groupconsisting of a bond and C₁₋₂₀ alkylene; when L¹¹ is C₂₋₂₀ alkylene, oneor more non-adjacent CH₂ groups are optionally and independentlyreplaced with O, S, or NH; when L¹¹ is C₃₋₂₀ alkylene, one or more pairsof adjacent CH₂ groups are optionally and independently replaced withC(O)O or C(O)NH; R¹² is selected from the group consisting of H, C₁₋₁₈alkyl, C₁₋₁₈ haloalkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, (C₃₋₁₀cycloalkyl)-L¹²-, (C₆₋₁₀ aryl)-L¹²-, (5- to 10-membered heteroaryl) (5-to 10-membered heterocyclyl)-L¹²-, cyano, halo, nitro, N(R¹⁸)₂, B(R¹⁹)₂,Si(R¹⁹)₃, C(O)OR¹⁷, C(O)SR¹⁷, C(O)N(R¹⁷)₂, C(O)R¹⁷, C(O)ON(R¹⁷)₂,C(O)NR¹⁷OR¹⁸, C(O)C(O)OR¹⁷, and P(O)(OR¹⁷)₂; C₁₋₁₈ alkyl, C₃₋₁₀cycloalkyl, C₆₋₁₀ aryl, 5- to 10-membered heteroaryl, and 5- to10-membered heterocyclyl in R¹² are optionally and independentlysubstituted with one or more R^(12a); each R^(12a) is independentlyselected from the group consisting of halogen, cyano, —OH, C₁₋₆ alkyl,C₁₋₆ alkoxy, C₁₋₆ heteroalkyl, C₂₋₆ alkynyl, C₃₋₁₀ cycloalkyl, C₆₋₁₀aryl, and 5- to 10-membered heterocyclyl; L¹² is selected from the groupconsisting of a bond and C₁₋₂₀ alkylene; when L¹² is C₂₋₂₀ alkylene, oneor more non-adjacent CH₂ groups are optionally and independentlyreplaced with O, S, or NH; when L¹² is C₃₋₂₀ alkylene, one or more pairsof adjacent CH₂ groups are optionally and independently replaced withC(O)O or C(O)NH; R¹³ and R¹⁴ are independently selected from the groupconsisting of H, optionally substituted C₁₋₁₈ alkyl, optionallysubstituted C₁₋₁₈ alkoxy, C₁₋₁₈ haloalkyl (e.g., C₁₋₁₈ polyfluoroalkyl),C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl,optionally substituted 5- to 10-membered heteroaryl, optionallysubstituted 5- to 10-membered heterocyclyl, cyano, halo, nitro, N(R¹⁸)₂,B(R¹⁹)₂, Si(R¹⁹)₃, C(O)OR¹⁷, C(O)SR¹⁷, C(O)N(R¹⁷)₂, C(O)R¹⁷,C(O)ON(R¹⁷)₂, C(O)NR¹⁷OR¹⁸, C(O)C(O)OR¹⁷ and P(O)(OR¹⁷)₂; and each R¹⁷,R¹⁸, and R¹⁹ is independently selected from the group consisting of H,optionally substituted C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl,optionally substituted C₆₋₁₀ aryl, optionally substituted 5- to10-membered heteroaryl, and optionally substituted 5- to 10-memberedheterocyclyl. 55-63. (canceled)
 64. A cytochrome P450_(BM3) variantcomprising the amino acid sequence set forth in SEQ ID NO:3 and furthercomprising: i) a V78F mutation and a S438 mutation, or ii) a V78Ymutation.
 65. The cytochrome P450_(BM3) variant of claim 64, wherein thevariant comprises the V78F mutation and wherein the S438 mutation is anS438A mutation.
 66. (canceled)
 67. The cytochrome P450_(BM3) variant ofclaim 64, wherein the variant comprises the V78Y mutation and furthercomprises an S438 mutation.
 68. The cytochrome P450_(BM3) variant ofclaim 67, wherein the S438 mutation is an S438A mutation.
 69. Acytochrome P450_(BM3) variant comprising the amino acid sequence setforth in SEQ ID NO:2 and further comprising: i) an A87F mutation and anF261 mutation or a T327 mutation, or ii) an A87W mutation.
 70. Thecytochrome P450_(BM3) variant of claim 69, wherein the variant comprisesthe A87F mutation and the F261 mutation or the T327 mutation, andfurther comprises one or more mutations selected from the groupconsisting of an S72 mutation, an A78 mutation, a an L188 mutation, aT269 mutation, an A330 mutation, a T436 mutation, and an L437 mutation.71. (canceled)
 72. The cytochrome P450_(BM3) variant of claim 69,wherein the variant comprises the A87W mutation and further comprisesone or mutations selected from the group consisting of an F261 mutation,a T327 mutation, a V328 mutation, a T436 mutation, and an L437 mutation.